Scintillator nanocrystal-containing compositions and methods for their use

ABSTRACT

There are provided, inter alia, compositions including a scintillator nanocrystal linked to a chemical agent moiety through a scintillator-activated photocleavable linker, and methods of use thereof.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/052,526 filed Feb. 24, 2016, issued as U.S. Pat. No. 10,864,272,which is a US National Stage under 35 USC § 371 of InternationalApplication PCT/US2014/053487 filed Aug. 29, 2014, which claims priorityto U.S. Application No. 61/872,122 filed Aug. 30, 2013, each of which isincorporated herein by reference in its entirety and for all purposes.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH AND DEVELOPMENT

This invention was made with Government support under grant No. CA187528awarded by the National Institutes of Health. The Government has certainrights in this invention.

BACKGROUND OF THE INVENTION

For decades medical radiation specialists have sought to activate bylocal radiation beams, a nontoxic, inactive version of a cancer drug,(e.g., a prodrug) selectively at cancers and not body tissues ingeneral. This strategy is attractive because it aims to overwhelm tumorresistance mechanisms by allowing high drug concentrations at tumorfoci, while sparing normal tissues and organs from toxicity, andreducing the generally damaging radiation doses needed to control tumorburden. Drug activity focused on areas adjacent to tumors that woulddestroy the micro metastases that are so challenging to selectivelyexcise or treat would be desirable. Single cell infiltration thatsignificantly diminishes by ablating the active margin of primary andsecondary tumors, especially in early disease stages, would also bedesirable.

Solutions to these and other issues are provided herein.

BRIEF SUMMARY OF THE INVENTION

There is disclosed herein, inter alia, the novel concept of completelybypassing oxygen quenching by linking a prodrug to a nanocrystalradiation scintillator. See e.g., FIG. 1 and Scheme 1. For example,embodiments are provided herein in which a drug is inactive while linkedto the crystal, but in response to radiation the scintillator emitslight to break the chemical linker, thereby releasing active drug.Without wishing to be bound by any theory, one rationale underlying thisdisclosure is that it makes feasible a transformative process for thewidespread development of radiation activatable therapeutics. Indeed,embodiments provided herein offer an entirely new pharmacologic approachbased on externally controlled, localized drug activation/release.Moreover, embodiments provided herein will work against many localizeddiseases like localized infections. For example, intravenously injectednanoparticles may concentrate at tumor foci by leaking through typicallyincomplete tumor vessels, by adhering to tumor microvessels viawell-established targeting ligands, and by penetration of the bloodbrain barrier (BBB) both passively and actively via transferrin ligands.Thousands of drug molecules may be linked to a single 100-150 nmscintillator crystal, and millions of such crystals can be injected.Indeed, 100 nm liposomal nanoparticles can deliver sufficient drug tocompletely suppress tumor growth in experimental animal models. Inembodiments, with the drug molecules bound very closely to thescintillator, radiation-induced drug release can occur with highefficiency.

In a first aspect, there is provided a composition including ascintillator nanocrystal linked to a chemical agent moiety through ascintillator-activated photocleavable linker.

In another aspect, there is provided a method for delivering a chemicalagent moiety to a target site. The method includes: (i) providing acomposition including a scintillator nanocrystal linked to a chemicalagent moiety through scintillator-activated photocleavable linker asdisclosed herein to a location at or near a target site and (ii)cleaving the chemical agent from the remainder of the composition byexposing the composition to radiation thereby delivering the chemicalagent to the target site. The term “target site” and the like refer, inthe usual and customary sense, to a site which can benefit by localadministration of the chemical agent moiety. In medical applications,the chemical agent moiety can be a drug moiety, a hormone moiety or adetectable moiety.

In another aspect, there is provided a method of delivering a chemicalagent moiety to a subject. The method includes: (i) administering acomposition including a scintillator nanocrystal linked to a chemicalagent moiety through scintillator-activated photocleavable linker asdisclosed herein to the subject, and (ii) cleaving the chemical agentfrom the remainder of the compound by exposing the composition toradiation thereby delivering the chemical agent to the subject.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts an exemplary cartoon design scheme for scintillatornanocrystal coated with lipid bilayer and attached to doxorubicin (“Dox”or “Doxo”) via a photocleavable linker. The scintillator nanocrystal maybe on the order of 100 nm in diameter, and total diameter ≈100-150 nm.Legend: 101: scintillator core; 102: inert hydrophilic shell; 103:external lipid bilayer; 104: expanded view of external lipid bilayer,indicated by arrow from 103, and adherent to inert hydrophilic shell;105: photocleavable linker showing attachment to exemplary lipid bilayer106 and to chemical agent moiety (e.g., Dox); 106: expanded view ofexternal lipid bilayer, indicated by arrow from 104, e.g., DOPE neutrallipid. Element 106 can be conjugated with chemical agent moiety, e.g.,drug (e.g., Dox), polyethylene glycol (PEG), detectable label (e.g.,fluorescent label), or other chemical agent moiety disclosed herein. Thephotocleavable linker can cleave under exposure to UV light (e.g., about350-360 nm).

FIG. 2 depicts light activation scheme using a linker, as exemplified inFIG. 1. Cytotoxic Dox is shown below on the left after UV lightexposure.

FIG. 3 depicts radioluminescence spectra and transmission electronmicrograph of BaF₂:Ce³⁺. Inset: transmission electron micrograph ofsingle core BaF₂:Ce³⁺ prepared by co-precipitation.

FIG. 4 depicts photoluminescence (PL) emission spectra of Y₂O₃:Eu³⁺ andY₂O₃:Eu³⁺/SiO₂ core shell particles. Inset: transmission electronmicrograph of the core/shell.

FIG. 5 depicts an exemplary design scheme for the synthesis of a dual,a5B1/avb3 ligand, and conjugation of this ligand to DOPE lipid. Thetable notation (ICSO) indicates that biochemical binding of this ligandto both integrins is very effective, i.e., low nanomolar range.

FIG. 6 depicts energy levels in Pr³⁺ ion showing the electronictransitions. The 4f5d (1) and 4f5d(2) levels are boxed.

FIG. 7 depicts representative X-ray diffraction (XRD) pattern of theYAG-Pr (1 at. %) obtained by combustion synthesis and post-annealed at1200° C. for 2 hr.

FIG. 8 depicts Transmission Electron Micrograph (TEM) image of theUV-emitting nanophosphor powder (Y_(1-x)Pr_(x))₃Al₅O₁₂ (x=1.0 at. % Pr)analyzed at 200 kV.

FIG. 9 depicts TEM images of representative samples of thedeagglomerated nanophosphors (Y_(1-x)Pr_(x))₃Al₅O₂ after ultrasonicationprocessing of 10 min.

FIG. 10 depicts photoluminescence spectra of (Y_(1-x)Pr_(x))₃Al₅O₁₂ withdifferent Pr concentration of x=0.5, 1.0, 1.5 at. %. The excitationwavelength for all Pr-doping concentrations was λ_(exc)=292 nm.

FIG. 11 depicts radioluminescence emission spectra of(Y_(1-x)Pr_(x))₃Al₅O₁₂ with different Pr concentration of x=0.5, 1.0,1.5, 1.75 at. %. under X-ray excitation of 50 KeV.

FIG. 12. X-ray diffraction patterns recorded for(Lu_(1-α-β)Y_(α)Pr_(ρ))₂SiO₅ powders obtained by combustion synthesisand heated in air at 1200° C. (1) (Lu_(0.75)Y_(0.2)Pr_(0.05))₂SiO₅heated for 2 h, (2) (Lu_(0.75)Y_(0.2)Pr_(0.5))₂SiO₅ heated for 4 h and(3) (Lu_(0.505)Y_(0.49)Pr_(0.005))₂SiO₅ heated for 4 h. Principalindexed peaks correspond to the monoclinic phase Lu₂SiO₅ JCPDS 41-0239.Two residual phases (♦) Lu₂SiO₇ JCPDS 35-0326 and (●) Lu₂O₃ JCPDS12-0728 can be also observed in some of the patterns.

FIG. 13. TEM micrograph of the morphology observed in(Lu_(0.505)Y_(0.49)Pr_(0.005))₂SiO₅ powders obtained by combustionsynthesis and then heated in air at 1200° C. for 6 h.

FIG. 14. Emission spectra recorded at room temperature of(Lu_(1-α-β)Y_(α)Pr_(β))₂SiO₅ powders obtained by combustion synthesisand heated in air at 1200° C. (i) (Lu_(0.75)Y_(0.2)Pr_(0.05))₂SiO₅heated for 2 h, (ii) (Lu_(0.75)Y_(0.2)Pr_(0.05))₂SiO₅ heated for 4 h and(iii) (Lu_(0.505)Y_(0.49)Pr_(0.005))₂SiO₅ heated for 4 h. Maximumemissions peaks centered at λ=280 nm and λ=315 nm, corresponding to thePr³⁺ forbidden transitions ⁵D₂→4f (³P₂) and ⁵D₁→4 f (³H₄), respectively.

FIG. 15. Pr³⁺ electronic transitions in insulator host lattice.

FIGS. 16A-16B. Spectral scintillation decays of samples and heated inair at 1200° C. collected at room temperature (λ_(ex)=252 nm). (FIG.16A) (Lu_(0.75)Y_(0.2)Pr_(0.05))₂SiO₅ heated for 2-h; (FIG. 16B)(Lu_(0.75)Y_(0.2)Pr_(0.05))₂SiO₅ heated for 4 h.

FIGS. 17A-17D. Thermoluminescence (TL) fading curve of YAG nanophosphorsdoped with 0.5 at. % (FIG. 17A), 1.0 at. % (FIG. 17B), 1.5 at. % (FIG.17C) and 2.0 at. % (FIG. 17D) of Pr³⁺ in a period of 24 hr. Theirradiation dose was 20 Gy.

FIG. 18. Thermoluminescence fading of YAG-Pr³⁺ nanophosphors irradiatedwith β-dose of 1 Gy as a function of time (hr). Legend: Pr⁺³ (at. %):0.5 (filled box); 1.0 (filled circle); 1.5 (triangle tip up); 2.0(triangle tip down).

FIG. 19. Thermoluminescence beta dosimetry of YAG-Pr³⁺ in the range of0-20 Gy. Legend: see FIG. 18.

FIGS. 20A-20D. Typical afterglow curves in the range of 1 to 500 Gy forall Pr³⁺ concentrations: (FIG. 20A) 0.5 at. %; (FIG. 20B) 1.0 at. %;(FIG. 20C) 1.5 at. %; and (FIG. 20D) 2.0 at. %.

FIG. 21. Afterglow intensity for several Pr³⁺ concentrations. Legend:see FIG. 18.

FIG. 22. Integrated radioluminescence emission intensity between 300-400nm of the nanoscintillators normalized to nanocrystalline Bi₄Ge₃O₁₂.CR=combustion reaction, SP=spray pyrolysis. 50 KeV x-rays, short pulse.

FIG. 23. Radioluminescence spectra emission intensity versus emissionwavelength. Peak intensity of emitted light in the range 250-750 nm wasdetermined for the indicated nanoscintillators.

FIGS. 24A-24B. Release of Dox with radiation dose. Dox was attached to ananoscintillator via a photocleavable linker which was in turn attachedto streptavidin which coated the nanocrystal. The system responded toradiation by release of Dox. FIG. 24A depicts a histogram showing resultafter 1 hours of dialysis after irradiation, so only the bound Doxremains; a stepwise response to radiation dose is observed. Legend: foreach radiation level (Non-irradiated, 2 gy, 4 gy), the left histogramentry represent Dox absorption in the dialysate, and the right entry isafter 1-hr of dialysis. FIG. 24B: After more complete dialysis, 24hours, it is observed that little bound Dox remains, and there is stilla dose response. Legend: for each radiation level (Non-irradiated, 2 gy,4 gy), the left histogram entry (open rectangle) represent Doxabsorption in the dialysate, and the right entry (closed rectangle) isafter 24-hr of dialysis.

FIGS. 25A-25C. Figures depict photomicrographs of accumulation ofnanoparticles at tumors with tumor regression. FIG. 25A: Image ofliposomal nanoparticles labeled with BODIPY inside tumor vessels.Liposomes are also permeating from the tumor vessels (arrow). FIGS.25B-25C: Figures depict in vivo imaging (rodent dorsal skinfold chamber)of disintegration of human M21L melanoma angiogenic vessels at Day 0(FIG. 25B) and after six days (FIG. 25C) of intravenous liposomaldoxorubicin. (tumor is αvβ3−, vessels are +ve). At Day 0 (FIG. 25B),extended vessels are observed. At Day 6 (FIG. 25C), there are observedloss of tips, vessels which are no longer extended, and loss of tumormass. Pruning of vessels caused a marked reduction in tumor size.

DETAILED DESCRIPTION Definitions

The abbreviations used herein have their conventional meaning within thechemical and biological arts. The chemical structures and formulae setforth herein are constructed according to the standard rules of chemicalvalency known in the chemical arts.

Where substituent groups are specified by their conventional chemicalformulae, written from left to right, they equally encompass thechemically identical substituents that would result from writing thestructure from right to left, e.g., —CH₂O— is equivalent to —OCH₂—.

The term “alkyl,” by itself or as part of another substituent, means,unless otherwise stated, a straight (i.e., unbranched) or branchedcarbon chain (or carbon), or combination thereof, which may be fullysaturated, mono- or polyunsaturated and can include mono-, di- andmultivalent radicals, having the number of carbon atoms designated(i.e., C₁-C₁₀ means one to ten carbons). Alkyl is an uncyclized chain.Examples of saturated hydrocarbon radicals include, but are not limitedto, groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl,isobutyl, sec-butyl, (cyclohexyl)methyl, homologs and isomers of, forexample, n-pentyl, n-hexyl, n-heptyl, n-octyl, and the like. Anunsaturated alkyl group is one having one or more double bonds or triplebonds. Examples of unsaturated alkyl groups include, but are not limitedto, vinyl, 2-propenyl, crotyl, 2-isopentenyl, 2-(butadienyl),2,4-pentadienyl, 3-(1,4-pentadienyl), ethynyl, 1- and 3-propynyl,3-butynyl, and the higher homologs and isomers. An alkoxy is an alkylattached to the remainder of the molecule via an oxygen linker (—O—).

The term “alkylene,” by itself or as part of another substituent, means,unless otherwise stated, a divalent radical derived from an alkyl, asexemplified, but not limited by, —CH₂CH₂CH₂CH₂—. Typically, an alkyl (oralkylene) group will have from 1 to 24 carbon atoms, with those groupshaving 10 or fewer carbon atoms being preferred for the compositions andmethods disclosed herein. A “lower alkyl” or “lower alkylene” is ashorter chain alkyl or alkylene group, generally having eight or fewercarbon atoms. The term “alkenylene,” by itself or as part of anothersubstituent, means, unless otherwise stated, a divalent radical derivedfrom an alkene.

The term “heteroalkyl” by itself or in combination with another termmeans, unless otherwise stated, a stable straight or branched chain, orcombinations thereof, including at least one carbon atom and at leastone heteroatom selected from the group consisting of O, N, P, Si, and S,and wherein the nitrogen and sulfur atoms may optionally be oxidized,and the nitrogen heteroatom may optionally be quaternized. Theheteroatom(s) O, N, P, S, B, As, and Si may be placed at any interiorposition of the heteroalkyl group or at the position at which the alkylgroup is attached to the remainder of the molecule. Heteroalkyl is anuncyclized chain. Examples include, but are not limited to:—CH₂—CH₂—O—CH₃, —CH₂—CH₂—NH—CH₃, —CH₂—CH₂—N(CH₃)—CH₃, —CH₂—S—CH₂—CH₃,—CH₂—CH₂, —S(O)—CH₃, —CH₂—CH₂—S(O)₂—CH₃, —CH═CH—O—CH₃, —Si(CH₃)₃,—CH₂—CH═N—OCH₃, —CH═CH—N(CH₃)—CH₃, —O—CH₃, —O—CH₂—CH₃, and —CN. Up totwo or three heteroatoms may be consecutive, such as, for example,—CH₂—NH—OCH₃ and —CH₂—O—Si(CH₃)₃.

Similarly, the term “heteroalkylene,” by itself or as part of anothersubstituent, means, unless otherwise stated, a divalent radical derivedfrom heteroalkyl, as exemplified, but not limited by,—CH₂—CH₂—S—CH₂—CH₂— and —CH₂—S—CH₂—CH₂—NH—CH₂—. For heteroalkylenegroups, heteroatoms can also occupy either or both of the chain termini(e.g., alkyleneoxy, alkylenedioxy, alkyleneamino, alkylenediamino, andthe like). Still further, for alkylene and heteroalkylene linkinggroups, no orientation of the linking group is implied by the directionin which the formula of the linking group is written. For example, theformula —C(O)₂R′— represents both —C(O)₂R′— and —R′C(O)₂—. As describedabove, heteroalkyl groups, as used herein, include those groups that areattached to the remainder of the molecule through a heteroatom, such as—C(O)R′, —C(O)NR′, —NR′R″, —OR′, —SR′, and/or —SO₂R′. Where“heteroalkyl” is recited, followed by recitations of specificheteroalkyl groups, such as —NR′R″ or the like, it will be understoodthat the terms heteroalkyl and —NR′R″ are not redundant or mutuallyexclusive. Rather, the specific heteroalkyl groups are recited to addclarity. Thus, the term “heteroalkyl” should not be interpreted hereinas excluding specific heteroalkyl groups, such as —NR′R″ or the like.

The terms “cycloalkyl” and “heterocycloalkyl,” by themselves or incombination with other terms, mean, unless otherwise stated, cyclicversions of “alkyl” and “heteroalkyl,” respectively. Cycloalkyl andheteroalkyl are not aromatic. Additionally, for heterocycloalkyl, aheteroatom can occupy the position at which the heterocycle is attachedto the remainder of the molecule. Examples of cycloalkyl include, butare not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, and the like. Examples ofheterocycloalkyl include, but are not limited to,1-(1,2,5,6-tetrahydropyridyl), 1-piperidinyl, 2-piperidinyl,3-piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl,tetrahydrofuran-3-yl, tetrahydrothien-2-yl, tetrahydrothien-3-yl,1-piperazinyl, 2-piperazinyl, and the like. A “cycloalkylene” and a“heterocycloalkylene,” alone or as part of another substituent, means adivalent radical derived from a cycloalkyl and heterocycloalkyl,respectively.

The terms “halo” or “halogen,” by themselves or as part of anothersubstituent, mean, unless otherwise stated, a fluorine, chlorine,bromine, or iodine atom. Additionally, terms such as “haloalkyl” aremeant to include monohaloalkyl and polyhaloalkyl. For example, the term“halo(C₁-C₄)alkyl” includes, but is not limited to, fluoromethyl,difluoromethyl, trifluoromethyl, 2,2,2-trifluoroethyl, 4-chlorobutyl,3-bromopropyl, and the like.

The term “acyl” means, unless otherwise stated, —C(O)R where R is asubstituted or unsubstituted alkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heteroalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl, orsubstituted or unsubstituted heteroaryl.

The term “aryl” means, unless otherwise stated, a polyunsaturated,aromatic, hydrocarbon substituent, which can be a single ring ormultiple rings (preferably from 1 to 3 rings) that are fused together(i.e., a fused ring aryl) or linked covalently. A fused ring aryl refersto multiple rings fused together wherein at least one of the fused ringsis an aryl ring. The term “heteroaryl” refers to aryl groups (or rings)that contain at least one heteroatom such as N, O, or S, wherein thenitrogen and sulfur atoms are optionally oxidized, and the nitrogenatom(s) are optionally quaternized. Thus, the term “heteroaryl” includesfused ring heteroaryl groups (i.e., multiple rings fused togetherwherein at least one of the fused rings is a heteroaromatic ring). A5,6-fused ring heteroarylene refers to two rings fused together, whereinone ring has 5 members and the other ring has 6 members, and wherein atleast one ring is a heteroaryl ring. Likewise, a 6,6-fused ringheteroarylene refers to two rings fused together, wherein one ring has 6members and the other ring has 6 members, and wherein at least one ringis a heteroaryl ring. And a 6,5-fused ring heteroarylene refers to tworings fused together, wherein one ring has 6 members and the other ringhas 5 members, and wherein at least one ring is a heteroaryl ring. Aheteroaryl group can be attached to the remainder of the moleculethrough a carbon or heteroatom. Non-limiting examples of aryl andheteroaryl groups include phenyl, naphthyl, pyrrolyl, pyrazolyl,pyridazinyl, triazinyl, pyrimidinyl, imidazolyl, pyrazinyl, purinyl,oxazolyl, isoxazolyl, thiazolyl, furyl, thienyl, pyridyl, pyrimidyl,benzothiazolyl, benzoxazoyl benzimidazolyl, benzofuran, isobenzofuranyl,indolyl, isoindolyl, benzothiophenyl, isoquinolyl, quinoxalinyl,quinolyl, 1-naphthyl, 2-naphthyl, 4-biphenyl, 1-pyrrolyl, 2-pyrrolyl,3-pyrrolyl, 3-pyrazolyl, 2-imidazolyl, 4-imidazolyl, pyrazinyl,2-oxazolyl, 4-oxazolyl, 2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl,4-isoxazolyl, 5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl,2-furyl, 3-furyl, 2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl,2-pyrimidyl, 4-pyrimidyl, 5-benzothiazolyl, purinyl, 2-benzimidazolyl,5-indolyl, 1-isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl,3-quinolyl, and 6-quinolyl. Substituents for each of the above notedaryl and heteroaryl ring systems are selected from the group ofacceptable substituents described below. An “arylene” and a“heteroarylene,” alone or as part of another substituent, mean adivalent radical derived from an aryl and heteroaryl, respectively. Aheteroaryl group substituent may be a —O-bonded to a ring heteroatomnitrogen.

A “fused ring aryl-heterocycloalkyl” is an aryl fused to aheterocycloalkyl. A “fused ring heteroaryl-heterocycloalkyl” is aheteroaryl fused to a heterocycloalkyl. A “fused ringheterocycloalkyl-cycloalkyl” is a heterocycloalkyl fused to acycloalkyl. A “fused ring heterocycloalkyl-heterocycloalkyl” is aheterocycloalkyl fused to another heterocycloalkyl. Fused ringaryl-heterocycloalkyl, fused ring heteroaryl-heterocycloalkyl, fusedring heterocycloalkyl-cycloalkyl, or fused ringheterocycloalkyl-heterocycloalkyl may each independently beunsubstituted or substituted with one or more of the substituentsdescribed herein. Fused ring aryl-heterocycloalkyl, fused ringheteroaryl-heterocycloalkyl, fused ring heterocycloalkyl-cycloalkyl, orfused ring heterocycloalkyl-heterocycloalkyl may each independently benamed according to the size of each of the fused rings. Thus, forexample, 6,5 aryl-heterocycloalkyl fused ring describes a 6 memberedaryl moiety fused to a 5 membered heterocycloalkyl. Spirocyclic ringsare two or more rings wherein adjacent rings are attached through asingle atom. The individual rings within spirocyclic rings may beidentical or different. Individual rings in spirocyclic rings may besubstituted or unsubstituted and may have different substituents fromother individual rings within a set of spirocyclic rings. Possiblesubstituents for individual rings within spirocyclic rings are thepossible substituents for the same ring when not part of spirocyclicrings (e.g., substituents for cycloalkyl or heterocycloalkyl rings).Spirocylic rings may be substituted or unsubstituted cycloalkyl,substituted or unsubstituted cycloalkylene, substituted or unsubstitutedheterocycloalkyl or substituted or unsubstituted heterocycloalkylene andindividual rings within a spirocyclic ring group may be any of theimmediately previous list, including having all rings of one type (e.g.,all rings being substituted heterocycloalkylene wherein each ring may bethe same or different substituted heterocycloalkylene). When referringto a spirocyclic ring system, heterocyclic spirocyclic rings means aspirocyclic rings wherein at least one ring is a heterocyclic ring andwherein each ring may be a different ring. When referring to aspirocyclic ring system, substituted spirocyclic rings means that atleast one ring is substituted and each substituent may optionally bedifferent.

The term “oxo,” as used herein, means an oxygen that is double bonded toa carbon atom.

Each of the above terms (e.g., “alkyl,” “heteroalkyl,” “aryl,” and“heteroaryl”) includes both substituted and unsubstituted forms of theindicated radical. Preferred substituents for each type of radical areprovided below.

Substituents for the alkyl and heteroalkyl radicals (including thosegroups often referred to as alkylene, alkenyl, heteroalkylene,heteroalkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, andheterocycloalkenyl) can be one or more of a variety of groups selectedfrom, but not limited to, —OR′, ═O, ═NR′, ═N—OR′, —NR′R″, —SR′,-halogen, —SiR′R″R′″, —OC(O)R′, —C(O)R′, —CO₂R′, —CONR′R″, —OC(O)NR′R″,—NR″C(O)R′, —NR′—C(O)NR″R′″, —NR″C(O)₂R′, —NR—C(NR′R″R′″)═NR″″,—NR—C(NR′R″)═NR′″, —S(O)R′, —S(O)₂R′, —S(O)₂NR′R″, —NRSO₂R′, —NR′NR″R′″,—ONR′R″, —NR′C═(O)NR″NR′″R″″, —CN, —NO₂, —NR′SO₂R″, —NR′C═(O)R″,—NR′C(O)—OR″, —NR′OR″, in a number ranging from zero to (2m′+1), wherem′ is the total number of carbon atoms in such radical. R, R′, R″, R′″,and R″″ each preferably independently refer to hydrogen, substituted orunsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl (e.g., aryl substituted with 1-3 halogens),substituted or unsubstituted heteroaryl, substituted or unsubstitutedalkyl, alkoxy, or thioalkoxy groups, or arylalkyl groups. When acompound disclosed herein includes more than one R group, for example,each of the R groups is independently selected as are each R′, R″, R′″,and R″″ group when more than one of these groups is present. When R′ andR″ are attached to the same nitrogen atom, they can be combined with thenitrogen atom to form a 4-, 5-, 6-, or 7-membered ring. For example,—NR′R″ includes, but is not limited to, 1-pyrrolidinyl and4-morpholinyl. From the above discussion of substituents, one of skillin the art will understand that the term “alkyl” is meant to includegroups including carbon atoms bound to groups other than hydrogengroups, such as haloalkyl (e.g., —CF₃ and —CH₂CF₃) and acyl (e.g.,—C(O)CH₃, —C(O)CF₃, —C(O)CH₂OCH₃, and the like).

Similar to the substituents described for the alkyl radical,substituents for the aryl and heteroaryl groups are varied and areselected from, for example: —OR′, —NR′R″, —SR′, -halogen, —SiR′R″R′″,—OC(O)R′, —C(O)R′, —CO₂R′, —CONR′R″, —OC(O)NR′R″, —NR″C(O)R′,—NR′—C(O)NR″R′″, —NR″C(O)₂R′, —NR—C(NR′R″R′″)═NR″″, —NR—C(NR′R″)═NR′″,—S(O)R′, —S(O)₂R′, —S(O)₂NR′R″, —NRSO₂R′, —NR′NR″R′″, —ONR′R″,—NR′C═(O)NR″NR′″R″″, —CN, —NO₂, —R′, —N₃, —CH(Ph)₂, fluoro(C₁-C₄)alkoxy,and fluoro(C₁-C₄)alkyl, —NR′SO₂R″, —NR′C═(O)R″, —NR′C(O)—OR″, —NR′OR″,in a number ranging from zero to the total number of open valences onthe aromatic ring system; and where R′, R″, R′″, and R″″ are preferablyindependently selected from hydrogen, substituted or unsubstitutedalkyl, substituted or unsubstituted heteroalkyl, substituted orunsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl,substituted or unsubstituted aryl, and substituted or unsubstitutedheteroaryl. When a compound disclosed herein includes more than one Rgroup, for example, each of the R groups is independently selected asare each R′, R″, R′″, and R″″ groups when more than one of these groupsis present.

Substituents for rings (e.g., cycloalkyl, heterocycloalkyl, aryl,heteroaryl, cycloalkylene, heterocycloalkylene, arylene, orheteroarylene) may be depicted as substituents on the ring rather thanon a specific atom of a ring (commonly referred to as a floatingsubstituent). In such a case, the substituent may be attached to any ofthe ring atoms (obeying the rules of chemical valency) and in the caseof fused rings or spirocyclic rings, a substituent depicted asassociated with one member of the fused rings or spirocyclic rings (afloating substituent on a single ring), may be a substituent on any ofthe fused rings or spirocyclic rings (a floating substituent on multiplerings). When a substituent is attached to a ring, but not a specificatom (a floating substituent), and a subscript for the substituent is aninteger greater than one, the multiple substituents may be on the sameatom, same ring, different atoms, different fused rings, differentspirocyclic rings, and each substituent may optionally be different.Where a point of attachment of a ring to the remainder of a molecule isnot limited to a single atom (a floating substituent), the attachmentpoint may be any atom of the ring and in the case of a fused ring orspirocyclic ring, any atom of any of the fused rings or spirocyclicrings while obeying the rules of chemical valency. Where a ring, fusedrings, or spirocyclic rings contain one or more ring heteroatoms and thering, fused rings, or spirocyclic rings are shown with one more floatingsubstituents (including, but not limited to, points of attachment to theremainder of the molecule), the floating substituents may be bonded tothe heteroatoms. Where the ring heteroatoms are shown bound to one ormore hydrogens (e.g., a ring nitrogen with two bonds to ring atoms and athird bond to a hydrogen) in the structure or formula with the floatingsubstituent, when the heteroatom is bonded to the floating substituent,the substituent will be understood to replace the hydrogen, whileobeying the rules of chemical valency.

Two or more substituents may optionally be joined to form aryl,heteroaryl, cycloalkyl, or heterocycloalkyl groups. Such so-calledring-forming substituents are typically, though not necessarily, foundattached to a cyclic base structure. In one embodiment, the ring-formingsubstituents are attached to adjacent members of the base structure. Forexample, two ring-forming substituents attached to adjacent members of acyclic base structure create a fused ring structure. In anotherembodiment, the ring-forming substituents are attached to a singlemember of the base structure. For example, two ring-forming substituentsattached to a single member of a cyclic base structure create aspirocyclic structure. In yet another embodiment, the ring-formingsubstituents are attached to non-adjacent members of the base structure.

Two of the substituents on adjacent atoms of the aryl or heteroaryl ringmay optionally form a ring of the formula -T-C(O)—(CRR′)_(q)—U—, whereinT and U are independently —NR—, —O—, —CRR′—, or a single bond, and q isan integer of from 0 to 3. Alternatively, two of the substituents onadjacent atoms of the aryl or heteroaryl ring may optionally be replacedwith a substituent of the formula -A-(CH₂)_(r)—B—, wherein A and B areindependently —CRR′—, —O—, —NR—, —S—, —S(O)—, —S(O)₂—, —S(O)₂NR′—, or asingle bond, and r is an integer of from 1 to 4. One of the single bondsof the new ring so formed may optionally be replaced with a double bond.Alternatively, two of the substituents on adjacent atoms of the aryl orheteroaryl ring may optionally be replaced with a substituent of theformula —(CRR′)_(s)—X′—(C″R″R′″)_(d)—, where s and d are independentlyintegers of from 0 to 3, and X′ is —O—, —NR′—, —S—, —S(O)—, —S(O)₂—, or—S(O)₂NR′—. The substituents R, R′, R″, and R′″ are preferablyindependently selected from hydrogen, substituted or unsubstitutedalkyl, substituted or unsubstituted heteroalkyl, substituted orunsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl,substituted or unsubstituted aryl, and substituted or unsubstitutedheteroaryl.

As used herein, the terms “heteroatom” or “ring heteroatom” are meant toinclude, oxygen (O), nitrogen (N), sulfur (S), phosphorus (P), Boron(B), Arsenic (As), and silicon (Si).

A “substituent group,” as used herein, means a group selected from thefollowing moieties: (A) oxo, halogen, —CF₃, —CN, —OH, —NH₂, —COOH,—CONH₂, —NO₂, —SH, —S₂Cl, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂,—NHC═(O)NHNH₂, —NHC═(O) NH₂, —NHSO₂H, —NHC═(O)H, —NHC(O)—OH, —NHOH,—OCF₃, —OCHF₂, unsubstituted alkyl, unsubstituted heteroalkyl,unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstitutedaryl, unsubstituted heteroaryl, and (B) alkyl, heteroalkyl, cycloalkyl,heterocycloalkyl, aryl, and heteroaryl, substituted with at least onesubstituent selected from: (i) oxo, halogen, —CF₃, —CN, —OH, —NH₂,—COOH, —CONH₂, —NO₂, —SH, —SO₂Cl, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂,—NHC═(O)NHNH₂, —NHC═(O) NH₂, —NHSO₂H, —NHC═(O)H, —NHC(O)—OH, —NHOH,—OCF₃, —OCHF₂, unsubstituted alkyl, unsubstituted heteroalkyl,unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstitutedaryl, unsubstituted heteroaryl, and (ii) alkyl, heteroalkyl, cycloalkyl,heterocycloalkyl, aryl, and heteroaryl, substituted with at least onesubstituent selected from: (a) oxo, halogen, —CF₃, —CN, —OH, —NH₂,—COOH, —CONH₂, —NO₂, —SH, —SO₂Cl, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂,—NHC═(O)NHNH₂, —NHC═(O)NH₂, —NHSO₂H, —NHC═(O)H, —NHC(O)—OH, —NHOH,—OCF₃, —OCHF₂, unsubstituted alkyl, unsubstituted heteroalkyl,unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstitutedaryl, unsubstituted heteroaryl, and (b) alkyl, heteroalkyl, cycloalkyl,heterocycloalkyl, aryl, or heteroaryl, substituted with at least onesubstituent selected from: oxo, halogen, —CF₃, —CN, —OH, —NH₂, —COOH,—CONH₂, —NO₂, —SH, —SO₂Cl, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂,—NHC═(O)NHNH₂, —NHC═(O) NH₂, —NHSO₂H, —NHC═(O)H, —NHC(O)—OH, —NHOH,—OCF₃, —OCHF₂, unsubstituted alkyl, unsubstituted heteroalkyl,unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstitutedaryl, and unsubstituted heteroaryl.

A “size-limited substituent” or “size-limited substituent group,” asused herein, means a group selected from all of the substituentsdescribed above for a “substituent group,” wherein each substituted orunsubstituted alkyl is a substituted or unsubstituted C₁-C₂₀ alkyl, eachsubstituted or unsubstituted heteroalkyl is a substituted orunsubstituted 2 to 20 membered heteroalkyl, each substituted orunsubstituted cycloalkyl is a substituted or unsubstituted C₃-C₈cycloalkyl, and each substituted or unsubstituted heterocycloalkyl is asubstituted or unsubstituted 3 to 8 membered heterocycloalkyl.

A “lower substituent” or “lower substituent group,” as used herein,means a group selected from all of the substituents described above fora “substituent group,” wherein each substituted or unsubstituted alkylis a substituted or unsubstituted C₁-C₈ alkyl, each substituted orunsubstituted heteroalkyl is a substituted or unsubstituted 2 to 8membered heteroalkyl, each substituted or unsubstituted cycloalkyl is asubstituted or unsubstituted C₃-C₇ cycloalkyl, and each substituted orunsubstituted heterocycloalkyl is a substituted or unsubstituted 3 to 7membered heterocycloalkyl.

In some embodiments, each substituted group described in the compoundsherein is substituted with at least one substituent group. Morespecifically, in some embodiments, each substituted alkyl, substitutedheteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl,substituted aryl, substituted heteroaryl, substituted alkylene,substituted heteroalkylene, substituted cycloalkylene, substitutedheterocycloalkylene, substituted arylene, and/or substitutedheteroarylene described in the compounds herein are substituted with atleast one substituent group. In other embodiments, at least one or allof these groups are substituted with at least one size-limitedsubstituent group. In other embodiments, at least one or all of thesegroups are substituted with at least one lower substituent group.

In other embodiments of the compounds herein, each substituted orunsubstituted alkyl may be a substituted or unsubstituted C₁-C₂₀ alkyl,each substituted or unsubstituted heteroalkyl is a substituted orunsubstituted 2 to 20 membered heteroalkyl, each substituted orunsubstituted cycloalkyl is a substituted or unsubstituted C₃-C₈cycloalkyl, and/or each substituted or unsubstituted heterocycloalkyl isa substituted or unsubstituted 3 to 8 membered heterocycloalkyl. In someembodiments of the compounds herein, each substituted or unsubstitutedalkylene is a substituted or unsubstituted C₁-C₂₀ alkylene, eachsubstituted or unsubstituted heteroalkylene is a substituted orunsubstituted 2 to 20 membered heteroalkylene, each substituted orunsubstituted cycloalkylene is a substituted or unsubstituted C₃-C₈cycloalkylene, and/or each substituted or unsubstitutedheterocycloalkylene is a substituted or unsubstituted 3 to 8 memberedheterocycloalkylene.

In some embodiments, each substituted or unsubstituted alkyl is asubstituted or unsubstituted C₁-C₈ alkyl, each substituted orunsubstituted heteroalkyl is a substituted or unsubstituted 2 to 8membered heteroalkyl, each substituted or unsubstituted cycloalkyl is asubstituted or unsubstituted C₃-C₇ cycloalkyl, and/or each substitutedor unsubstituted heterocycloalkyl is a substituted or unsubstituted 3 to7 membered heterocycloalkyl. In some embodiments, each substituted orunsubstituted alkylene is a substituted or unsubstituted C₁-C₈ alkylene,each substituted or unsubstituted heteroalkylene is a substituted orunsubstituted 2 to 8 membered heteroalkylene, each substituted orunsubstituted cycloalkylene is a substituted or unsubstituted C₃-C₇cycloalkylene, and/or each substituted or unsubstitutedheterocycloalkylene is a substituted or unsubstituted 3 to 7 memberedheterocycloalkylene.

Certain compounds described herein possess asymmetric carbon atoms(optical or chiral centers) or double bonds; the enantiomers, racemates,diastereomers, tautomers, geometric isomers, stereoisometric forms thatmay be defined, in terms of absolute stereochemistry, as (R)- or (S)-or, as (D)- or (L)- for amino acids, and individual isomers areencompassed within the scope of the compounds disclosed herein. Thecompounds disclosed herein do not include those which are known in artto be too unstable to synthesize and/or isolate. The current disclosureis meant to include compounds in racemic and optically pure forms.Optically active (R)- and (S)-, or (D)- and (L)-isomers may be preparedusing chiral synthons or chiral reagents, or resolved using conventionaltechniques. When the compounds described herein contain olefinic bondsor other centers of geometric asymmetry, and unless specified otherwise,it is intended that the compounds include both E and Z geometricisomers.

As used herein, the term “isomers” refers to compounds having the samenumber and kind of atoms, and hence the same molecular weight, butdiffering in respect to the structural arrangement or configuration ofthe atoms.

The term “tautomer,” as used herein, refers to one of two or morestructural isomers which exist in equilibrium and which are readilyconverted from one isomeric form to another.

It will be apparent to one skilled in the art that certain compoundsdisclosed herein may exist in tautomeric forms, all such tautomericforms being within the scope of the compounds disclosed herein.

Unless otherwise stated, structures depicted herein are also meant toinclude all stereochemical forms of the structure; i.e., the (R) and (S)configurations for each asymmetric center. Therefore, singlestereochemical isomers as well as enantiomeric and diastereomericmixtures of the present compounds, generally recognized as stable bythose skilled in the art, are within the scope of the compoundsdisclosed herein.

Unless otherwise stated, structures depicted herein are also meant toinclude compounds which differ only in the presence of one or moreisotopically enriched atoms. For example, compounds having the presentstructures except for the replacement of a hydrogen by a deuterium ortritium, replacement of fluoride by ¹⁸F, or the replacement of a carbonby ¹³C- or ¹⁴C-enriched carbon are within the scope of the compoundsdisclosed herein.

The compounds disclosed herein may also contain unnatural proportions ofatomic isotopes at one or more of the atoms that constitute suchcompounds. For example, the compounds may be radiolabeled withradioactive isotopes, such as for example tritium (³H), fluoride (¹⁸F),iodine-125 (¹²⁵I), or carbon-14 (¹⁴C). All isotopic variations of thecompounds disclosed herein, whether radioactive or not, are encompassedwithin the scope of the compounds disclosed herein.

The symbol “

” denotes the point of attachment of a chemical moiety to the remainderof a molecule or chemical formula.

Where a moiety is substituted with an R substituent, the group may bereferred to as “R-substituted.” Where a moiety is R-substituted, themoiety is substituted with at least one R substituent and each Rsubstituent is optionally different. Where a particular R group ispresent in the description of a chemical genus (such as Formula (I)), aRoman decimal symbol may be used to distinguish each appearance of thatparticular R group. For example, where multiple R¹³ substituents arepresent, each R¹³ substituent may be distinguished as R^(13.1),R^(13.2), R^(13.3), R^(13.4), etc., wherein each of R^(13.1), R^(13.2),R^(13.3), R^(13.4), etc. is independently defined within the scope ofthe definition of R¹³ and optionally differently.

Description of compounds disclosed herein is limited by principles ofchemical bonding known to those skilled in the art. Accordingly, where agroup may be substituted by one or more of a number of substituents,such substitutions are selected so as to comply with principles ofchemical bonding and to give compounds which are not inherently unstableand/or would be known to one of ordinary skill in the art as likely tobe unstable under ambient conditions, such as aqueous, neutral, andseveral known physiological conditions. For example, a heterocycloalkylor heteroaryl is attached to the remainder of the molecule via a ringheteroatom in compliance with principles of chemical bonding known tothose skilled in the art thereby avoiding inherently unstable compounds.

“Analog,” or “analogue” are used in accordance with plain ordinarymeaning within Chemistry and Biology and refer to a chemical compoundthat is structurally similar to another compound (i.e., a so-called“reference” compound) but differs in composition, e.g., in thereplacement of one atom by an atom of a different element, or in thepresence of a particular functional group, or the replacement of onefunctional group by another functional group, or the absolutestereochemistry of one or more chiral centers of the reference compound.Accordingly, an analogue is a compound that is similar or comparable infunction and appearance but not in structure or origin to a referencecompound.

The term “pharmaceutically acceptable salts” is meant to include saltsof active compounds that are prepared with relatively nontoxic acids orbases, depending on the particular substituents found on the compoundsdescribed herein. When compounds disclosed herein contain relativelyacidic functionalities, base addition salts can be obtained bycontacting the neutral form of such compounds with a sufficient amountof the desired base, either neat or in a suitable inert solvent.Examples of pharmaceutically acceptable base addition salts includesodium, potassium, calcium, ammonium, organic amino, or magnesium salt,or a similar salt. When compounds disclosed herein contain relativelybasic functionalities, acid addition salts can be obtained by contactingthe neutral form of such compounds with a sufficient amount of thedesired acid, either neat or in a suitable inert solvent. Examples ofpharmaceutically acceptable acid addition salts include those derivedfrom inorganic acids like hydrochloric, hydrobromic, nitric, carbonic,monohydrogencarbonic, phosphoric, monohydrogenphosphoric,dihydrogenphosphoric, sulfuric, monohydrogensulfuric, hydriodic, orphosphorous acids and the like, as well as the salts derived fromrelatively nontoxic organic acids like acetic, propionic, isobutyric,maleic, malonic, benzoic, succinic, suberic, fumaric, lactic, mandelic,phthalic, benzenesulfonic, p-tolylsulfonic, citric, tartaric, oxalic,methanesulfonic, and the like. Also included are salts of amino acidssuch as arginate and the like, and salts of organic acids likeglucuronic or galactunoric acids and the like (see, for example, Bergeet al., “Pharmaceutical Salts”, Journal of Pharmaceutical Science, 1977,66, 1-19). Certain specific compounds disclosed herein contain bothbasic and acidic functionalities that allow the compounds to beconverted into either base or acid addition salts.

Thus, the compounds disclosed herein may exist as salts, such as withpharmaceutically acceptable acids. The compounds disclosed hereinincludes such salts. Examples of such salts include hydrochlorides,hydrobromides, sulfates, methanesulfonates, nitrates, maleates,acetates, citrates, fumarates, tartrates (e.g., (+)-tartrates,(−)-tartrates, or mixtures thereof including racemic mixtures),succinates, benzoates, and salts with amino acids such as glutamic acid.These salts may be prepared by methods known to those skilled in theart.

The neutral forms of the compounds are preferably regenerated bycontacting the salt with a base or acid and isolating the parentcompound in the conventional manner. The parent form of the compounddiffers from the various salt forms in certain physical properties, suchas solubility in polar solvents.

In addition to salt forms, there are provided compounds which are in aprodrug form. Prodrugs of the compounds described herein include thosecompounds that readily undergo chemical or enzymatic changes underphysiological conditions to provide the compounds disclosed herein.Additionally, prodrugs can be converted to the compounds disclosedherein by chemical or biochemical methods in an ex vivo environment. Forexample, prodrugs can be slowly converted to the compounds disclosedherein when placed in a transdermal patch reservoir with a suitableenzyme or chemical reagent.

Certain compounds disclosed herein can exist in unsolvated forms as wellas solvated forms, including hydrated forms. In general, the solvatedforms are equivalent to unsolvated forms and are encompassed within thescope disclosed herein. Certain compounds disclosed herein may exist inmultiple crystalline or amorphous forms. In general, all physical formsare equivalent for the uses disclosed herein and are intended to bewithin the scope of the compounds and methods disclosed herein.

As used herein, the term “salt” refers to acid or base salts of thecompounds used in the methods disclosed herein. Illustrative examples ofacceptable salts are mineral acid (hydrochloric acid, hydrobromic acid,phosphoric acid, and the like) salts, organic acid (acetic acid,propionic acid, glutamic acid, citric acid and the like) salts,quaternary ammonium (methyl iodide, ethyl iodide, and the like) salts.

The terms “treating”, or “treatment” refer to any indicia of success inthe treatment or amelioration of an injury, disease, pathology orcondition, including any objective or subjective parameter such asabatement; remission; diminishing of symptoms or making the injury,pathology or condition more tolerable to the patient; slowing in therate of degeneration or decline; making the final point of degenerationless debilitating; or improving a patient's physical or mentalwell-being. The treatment or amelioration of symptoms can be based onobjective or subjective parameters, including the results of a physicalexamination, neuropsychiatric exams, and/or a psychiatric evaluation.The term “treating” and conjugations thereof, include prevention of aninjury, pathology, condition, or disease.

An “effective amount” is an amount sufficient to accomplish a statedpurpose (e.g., achieve the effect for which it is administered, treat adisease, reduce enzyme activity, increase enzyme activity, reduce one ormore symptoms of a disease or condition). An example of an “effectiveamount” is an amount sufficient to contribute to the treatment,prevention, or reduction of a symptom or symptoms of a disease, whichcould also be referred to as a “therapeutically effective amount.” A“reduction” of a symptom or symptoms (and grammatical equivalents ofthis phrase) means decreasing of the severity or frequency of thesymptom(s), or elimination of the symptom(s). A “prophylacticallyeffective amount” of a drug is an amount of a drug that, whenadministered to a subject, will have the intended prophylactic effect,e.g., preventing or delaying the onset (or reoccurrence) of an injury,disease, pathology or condition, or reducing the likelihood of the onset(or reoccurrence) of an injury, disease, pathology, or condition, ortheir symptoms. The full prophylactic effect does not necessarily occurby administration of one dose, and may occur only after administrationof a series of doses. Thus, a prophylactically effective amount may beadministered in one or more administrations. The exact amounts willdepend on the purpose of the treatment, and will be ascertainable by oneskilled in the art using known techniques (see, e.g., Lieberman,Pharmaceutical Dosage Forms (vols. 1-3, 1992); Lloyd, The Art, Scienceand Technology of Pharmaceutical Compounding (1999); Pickar, DosageCalculations (1999); and Remington: The Science and Practice ofPharmacy, 20th Edition, 2003, Gennaro, Ed., Lippincott, Williams &Wilkins).

For any compound described herein, the therapeutically effective amountcan be initially determined from cell culture assays. Targetconcentrations will be those concentrations of active compound(s) thatare capable of achieving the methods described herein, as measured usingthe methods described herein or known in the art.

As is well known in the art, therapeutically effective amounts for usein humans can also be determined from animal models. For example, a dosefor humans can be formulated to achieve a concentration that has beenfound to be effective in animals. The dosage in humans can be adjustedby monitoring compounds effectiveness and adjusting the dosage upwardsor downwards, as described above. Adjusting the dose to achieve maximalefficacy in humans based on the methods described above and othermethods is well within the capabilities of the ordinarily skilledartisan.

Dosages may be varied depending upon the requirements of the patient andthe compound being employed. The dose administered to a patient, in thecontext of the methods disclosed herein should be sufficient to effect abeneficial therapeutic response in the patient over time. The size ofthe dose also will be determined by the existence, nature, and extent ofany adverse side-effects. Determination of the proper dosage for aparticular situation is within the skill of the practitioner. Generally,treatment is initiated with smaller dosages which are less than theoptimum dose of the compound. Thereafter, the dosage is increased bysmall increments until the optimum effect under circumstances isreached.

Dosage amounts and intervals can be adjusted individually to providelevels of the administered compound effective for the particularclinical indication being treated. This will provide a therapeuticregimen that is commensurate with the severity of the individual'sdisease state.

Utilizing the teachings provided herein, an effective prophylactic ortherapeutic treatment regimen can be planned that does not causesubstantial toxicity and yet is effective to treat the clinical symptomsdemonstrated by the particular patient. This planning should involve thecareful choice of active compound by considering factors such ascompound potency, relative bioavailability, patient body weight,presence and severity of adverse side effects, preferred mode ofadministration and the toxicity profile of the selected agent.

“Control” or “control experiment” is used in accordance with its plainordinary meaning and refers to an experiment in which the subjects orreagents of the experiment are treated as in a parallel experimentexcept for omission of a procedure, reagent, or variable of theexperiment. In some instances, the control is used as a standard ofcomparison in evaluating experimental effects. In embodiments, a controlis the measurement of the activity of a protein in the absence of acompound as described herein (including embodiments and examples).

“Contacting” is used in accordance with its plain ordinary meaning andrefers to the process of allowing at least two distinct species (e.g.,chemical compounds including biomolecules or cells) to becomesufficiently proximal to react, interact or physically touch. It shouldbe appreciated; however, the resulting reaction product can be produceddirectly from a reaction between the added reagents or from anintermediate from one or more of the added reagents which can beproduced in the reaction mixture.

The term “contacting” may include allowing two species to react,interact, or physically touch, wherein the two species may be a compoundas described herein and a protein or enzyme. Contacting may includeallowing a compound described herein to interact with a protein orenzyme that is involved in a signaling pathway.

As defined herein, the term “inhibition”, “inhibit”, “inhibiting” andthe like in reference to a protein-inhibitor interaction meansnegatively affecting (e.g., decreasing) the activity or function of theprotein relative to the activity or function of the protein in theabsence of the inhibitor. Inhibition may refer to reduction of a diseaseor symptoms of disease. Inhibition may refer to a reduction in theactivity of a particular protein or nucleic acid target. Thus,inhibition includes, at least in part, partially or totally blockingstimulation, decreasing, preventing, or delaying activation, orinactivating, desensitizing, or down-regulating signal transduction orenzymatic activity or the amount of a protein.

The term “modulator” refers to a composition that increases or decreasesthe level of a target molecule or the function of a target molecule orthe physical state of the target of the molecule.

The term “modulate” is used in accordance with its plain ordinarymeaning and refers to the act of changing or varying one or moreproperties. “Modulation” refers to the process of changing or varyingone or more properties. For example, a modulator of a target proteinchanges by increasing or decreasing a property or function of the targetmolecule or the amount of the target molecule. A modulator of a diseasedecreases a symptom, cause, or characteristic of the targeted disease.

“Selective” or “selectivity” or the like of a compound refers to thecompound's ability to discriminate between molecular targets.“Specific”, “specifically”, “specificity”, or the like of a compoundrefers to the compound's ability to cause a particular action, such asinhibition, to a particular molecular target with minimal or no actionto other proteins in the cell.

“Pharmaceutically acceptable excipient” and “pharmaceutically acceptablecarrier” refer to a substance that aids the administration of an activeagent to and absorption by a subject and can be included in thecompositions disclosed herein without causing a significant adversetoxicological effect on the patient. Non-limiting examples ofpharmaceutically acceptable excipients include water, NaCl, normalsaline solutions, lactated Ringer's, normal sucrose, normal glucose,binders, fillers, disintegrants, lubricants, coatings, sweeteners,flavors, salt solutions (such as Ringer's solution), alcohols, oils,gelatins, carbohydrates such as lactose, amylose or starch, fatty acidesters, hydroxymethycellulose, polyvinyl pyrrolidine, and colors, andthe like. Such preparations can be sterilized and, if desired, mixedwith auxiliary agents such as lubricants, preservatives, stabilizers,wetting agents, emulsifiers, salts for influencing osmotic pressure,buffers, coloring, and/or aromatic substances and the like that do notdeleteriously react with the compounds disclosed herein. One of skill inthe art will recognize that other pharmaceutical excipients are usefulin the compositions and methods disclosed herein.

The term “preparation” is intended to include the formulation of theactive compound with encapsulating material as a carrier providing acapsule in which the active component with or without other carriers, issurrounded by a carrier, which is thus in association with it.Similarly, cachets and lozenges are included. Tablets, powders,capsules, pills, cachets, and lozenges can be used as solid dosage formssuitable for oral administration.

As used herein, the term “administering” means oral administration,administration as a suppository, topical contact, intravenous,parenteral, intraperitoneal, intramuscular, intralesional, intrathecal,intranasal or subcutaneous administration, or the implantation of aslow-release device, e.g., a mini-osmotic pump, to a subject.Administration is by any route, including parenteral and transmucosal(e.g., buccal, sublingual, palatal, gingival, nasal, vaginal, rectal, ortransdermal). Parenteral administration includes, e.g., intravenous,intramuscular, intra-arteriole, intradermal, subcutaneous,intraperitoneal, intraventricular, and intracranial. Other modes ofdelivery include, but are not limited to, the use of liposomalformulations, intravenous infusion, transdermal patches, etc.

The compositions disclosed herein can be delivered by transdermally, bya topical route, formulated as applicator sticks, solutions,suspensions, emulsions, gels, creams, ointments, pastes, jellies,paints, powders, and aerosols. Oral preparations include tablets, pills,powder, dragees, capsules, liquids, lozenges, cachets, gels, syrups,slurries, suspensions, etc., suitable for ingestion by the patient.Solid form preparations include powders, tablets, pills, capsules,cachets, suppositories, and dispersible granules. Liquid formpreparations include solutions, suspensions, and emulsions, for example,water or water/propylene glycol solutions. The compositions disclosedherein may additionally include components to provide sustained releaseand/or comfort. Such components include high molecular weight, anionicmucomimetic polymers, gelling polysaccharides and finely-divided drugcarrier substrates. These components are discussed in greater detail inU.S. Pat. Nos. 4,911,920; 5,403,841; 5,212,162; and 4,861,760. Theentire contents of these patents are incorporated herein by reference intheir entirety for all purposes. The compositions disclosed herein canalso be delivered as microspheres for slow release in the body. Forexample, microspheres can be administered via intradermal injection ofdrug-containing microspheres, which slowly release subcutaneously (seeRao, J. Biomater Sci. Polym. Ed. 7:623-645, 1995; as biodegradable andinjectable gel formulations (see, e.g., Gao Pharm. Res. 12:857-863,1995); or, as microspheres for oral administration (see, e.g., Eyles, J.Pharm. Pharmacol. 49:669-674, 1997). In another embodiment, theformulations of the compositions disclosed herein can be delivered bythe use of liposomes which fuse with the cellular membrane or areendocytosed, i.e., by employing receptor ligands attached to theliposome, that bind to surface membrane protein receptors of the cellresulting in endocytosis. By using liposomes, particularly where theliposome surface carries receptor ligands specific for target cells, orare otherwise preferentially directed to a specific organ, one can focusthe delivery of the compositions disclosed herein into the target cellsin vivo. (See, e.g., Al-Muhammed, J. Microencapsul. 13:293-306, 1996;Chonn, Curr. Opin. Biotechnol. 6:698-708, 1995; Ostro, Am. J. Hosp.Pharm. 46:1576-1587, 1989). The compositions can also be delivered asnanoparticles.

Pharmaceutical compositions may include compositions wherein the activeingredient (e.g., compounds described herein, including embodiments orexamples) is contained in a therapeutically effective amount, i.e., inan amount effective to achieve its intended purpose. The actual amounteffective for a particular application will depend, inter alia, on thecondition being treated. When administered in methods to treat adisease, such compositions will contain an amount of active ingredienteffective to achieve the desired result, e.g., modulating the activityof a target molecule, and/or reducing, eliminating, or slowing theprogression of disease symptoms.

The dosage and frequency (single or multiple doses) administered to amammal can vary depending upon a variety of factors, for example,whether the mammal suffers from another disease, and its route ofadministration; size, age, sex, health, body weight, body mass index,and diet of the recipient; nature and extent of symptoms of the diseasebeing treated, kind of concurrent treatment, complications from thedisease being treated or other health-related problems. Othertherapeutic regimens or agents can be used in conjunction with themethods and compounds disclosed herein. Adjustment and manipulation ofestablished dosages (e.g., frequency and duration) are well within theability of those skilled in the art.

The compounds described herein can be used in combination with oneanother, with other active drugs known to be useful in treating adisease (e.g., anti-cancer drugs) or with adjunctive agents that may notbe effective alone, but may contribute to the efficacy of the activeagent. Thus, the compounds described herein may be co-administered withone another or with other active drugs known to be useful in treating adisease.

By “co-administer” it is meant that a compound described herein isadministered at the same time, just prior to, or just after theadministration of one or more additional therapies, for example, ananticancer agent as described herein. The compounds described herein canbe administered alone or can be co-administered to the patient.Co-administration is meant to include simultaneous or sequentialadministration of the compound individually or in combination (more thanone compound or agent). Thus, the preparations can also be combined,when desired, with other active substances (e.g., anticancer agents).

Co-administration includes administering one active agent (e.g., acomplex described herein) within 0.5, 1, 2, 4, 6, 8, 10, 12, 16, 20, or24 hours of a second active agent (e.g., anti-cancer agents). Alsocontemplated herein, are embodiments, where co-administration includesadministering one active agent within 0.5, 1, 2, 4, 6, 8, 10, 12, 16,20, or 24 hours of a second active agent. Co-administration includesadministering two active agents simultaneously, approximatelysimultaneously (e.g., within about 1, 5, 10, 15, 20, or 30 minutes ofeach other), or sequentially in any order. Co-administration can beaccomplished by co-formulation, i.e., preparing a single pharmaceuticalcomposition including both active agents. In other embodiments, theactive agents can be formulated separately. The active and/or adjunctiveagents may be linked or conjugated to one another. The compoundsdescribed herein may be combined with treatments for cancer such aschemotherapy or radiation therapy.

The term “associated” or “associated with” in the context of a substanceor substance activity or function associated with a disease means thatthe disease is caused by (in whole or in part), a symptom of the diseaseis caused by (in whole or in part) the substance or substance activityor function, or a side-effect of the compound (e.g., toxicity) is causedby (in whole or in part) the substance or substance activity orfunction.

“Patient,” “subject,” “patient in need thereof,” and “subject in needthereof” are herein used interchangeably and refer to a living organismsuffering from or prone to a disease or condition that can be treated byadministration of a pharmaceutical composition as provided herein.Non-limiting examples include humans, other mammals, bovines, rats,mice, dogs, monkeys, goat, sheep, cows, deer, and other non-mammaliananimals. In some embodiments, a patient is human. A “cancer-patient” isa patient suffering from, or prone to developing cancer.

“Disease,” “disorder” or “condition” refer to a state of being or healthstatus of a patient or subject capable of being treated with thecompounds or methods provided herein. Disease as used herein may referto cancer.

As used herein, the term “cancer” refers to all types of cancer,neoplasm, or malignant or benign tumors found in mammals, includingleukemia, carcinomas and sarcomas. Exemplary cancers include acutemyeloid leukemia (“AML”), chronic myelogenous leukemia (“CML”), andcancer of the brain, breast, pancreas, colon, liver, kidney, lung,non-small cell lung, melanoma, ovary, sarcoma, and prostate. Additionalexamples include, cervix cancers, stomach cancers, head & neck cancers,uterus cancers, mesothelioma, metastatic bone cancer, Medulloblastoma,Hodgkin's Disease, Non-Hodgkin's Lymphoma, multiple myeloma,neuroblastoma, ovarian cancer, rhabdomyosarcoma, primary thrombocytosis,primary macroglobulinemia, primary brain tumors, cancer, malignantpancreatic insulanoma, malignant carcinoid, urinary bladder cancer,premalignant skin lesions, testicular cancer, lymphomas, thyroid cancer,neuroblastoma, esophageal cancer, genitourinary tract cancer, malignanthypercalcemia, endometrial cancer, adrenal cortical cancer, andneoplasms of the endocrine and exocrine pancreas.

The term “leukemia” refers broadly to progressive, malignant diseases ofthe blood-forming organs and is generally characterized by a distortedproliferation and development of leukocytes and their precursors in theblood and bone marrow. Leukemia is generally clinically classified onthe basis of (1) the duration and character of the disease-acute orchronic; (2) the type of cell involved; myeloid (myelogenous), lymphoid(lymphogenous), or monocytic; and (3) the increase or non-increase inthe number abnormal cells in the blood-leukemic or aleukemic(subleukemic). The murine leukemia model is widely accepted as beingpredictive of in vivo anti-leukemic activity. It is believed that acompound that tests positive in the P388 cell assay will generallyexhibit some level of anti-leukemic activity regardless of the type ofleukemia being treated. Accordingly, the present disclosure includes amethod of treating leukemia, including treating acute myeloid leukemia,chronic lymphocytic leukemia, acute granulocytic leukemia, chronicgranulocytic leukemia, acute promyelocytic leukemia, adult T-cellleukemia, aleukemic leukemia, a leukocythemic leukemia, basophylicleukemia, blast cell leukemia, bovine leukemia, chronic myelocyticleukemia, leukemia cutis, embryonal leukemia, eosinophilic leukemia,Gross' leukemia, hairy-cell leukemia, hemoblastic leukemia,hemocytoblastic leukemia, histiocytic leukemia, stem cell leukemia,acute monocytic leukemia, leukopenic leukemia, lymphatic leukemia,lymphoblastic leukemia, lymphocytic leukemia, lymphogenous leukemia,lymphoid leukemia, lymphosarcoma cell leukemia, mast cell leukemia,megakaryocytic leukemia, micromyeloblastic leukemia, monocytic leukemia,myeloblastic leukemia, myelocytic leukemia, myeloid granulocyticleukemia, myelomonocytic leukemia, Naegeli leukemia, plasma cellleukemia, multiple myeloma, plasmacytic leukemia, promyelocyticleukemia, Rieder cell leukemia, Schilling's leukemia, stem cellleukemia, subleukemic leukemia, and undifferentiated cell leukemia.

The term “sarcoma” generally refers to a tumor which is made up of asubstance like the embryonic connective tissue and is generally composedof closely packed cells embedded in a fibrillar or homogeneoussubstance. Sarcomas which can be treated with a combination ofantineoplastic thiol-binding mitochondrial oxidant and an anticanceragent include a chondrosarcoma, fibrosarcoma, lymphosarcoma,melanosarcoma, myxosarcoma, osteosarcoma, Abemethy's sarcoma, adiposesarcoma, liposarcoma, alveolar soft part sarcoma, ameloblastic sarcoma,botryoid sarcoma, chloroma sarcoma, chorio carcinoma, embryonal sarcoma,Wilms' tumor sarcoma, endometrial sarcoma, stromal sarcoma, Ewing'ssarcoma, fascial sarcoma, fibroblastic sarcoma, giant cell sarcoma,granulocytic sarcoma, Hodgkin's sarcoma, idiopathic multiple pigmentedhemorrhagic sarcoma, immunoblastic sarcoma of B cells, lymphoma,immunoblastic sarcoma of T-cells, Jensen's sarcoma, Kaposi's sarcoma,Kupffer cell sarcoma, angiosarcoma, leukosarcoma, malignant mesenchymomasarcoma, parosteal sarcoma, reticulocytic sarcoma, Rous sarcoma,serocystic sarcoma, synovial sarcoma, and telangiectaltic sarcoma.

The term “melanoma” is taken to mean a tumor arising from themelanocytic system of the skin and other organs. Melanomas which can betreated with a combination of antineoplastic thiol-binding mitochondrialoxidant and an anticancer agent include, for example, acral-lentiginousmelanoma, amelanotic melanoma, benign juvenile melanoma, Cloudman'smelanoma, S91 melanoma, Harding-Passey melanoma, juvenile melanoma,lentigo maligna melanoma, malignant melanoma, nodular melanoma, subungalmelanoma, and superficial spreading melanoma.

The term “carcinoma” refers to a malignant new growth made up ofepithelial cells tending to infiltrate the surrounding tissues and giverise to metastases. Exemplary carcinomas which can be treated with acombination of antineoplastic thiol-binding mitochondrial oxidant and ananticancer agent include, for example, acinar carcinoma, acinouscarcinoma, adenocystic carcinoma, adenoid cystic carcinoma, carcinomaadenomatosum, carcinoma of adrenal cortex, alveolar carcinoma, alveolarcell carcinoma, basal cell carcinoma, carcinoma basocellulare, basaloidcarcinoma, basosquamous cell carcinoma, bronchioalveolar carcinoma,bronchiolar carcinoma, bronchogenic carcinoma, cerebriform carcinoma,cholangiocellular carcinoma, chorionic carcinoma, colloid carcinoma,comedo carcinoma, corpus carcinoma, cribriform carcinoma, carcinoma encuirasse, carcinoma cutaneum, cylindrical carcinoma, cylindrical cellcarcinoma, duct carcinoma, carcinoma durum, embryonal carcinoma,encephaloid carcinoma, epiermoid carcinoma, carcinoma epithelialeadenoides, exophytic carcinoma, carcinoma ex ulcere, carcinoma fibrosum,gelatiniforni carcinoma, gelatinous carcinoma, giant cell carcinoma,carcinoma gigantocellulare, glandular carcinoma, granulosa cellcarcinoma, hair-matrix carcinoma, hematoid carcinoma, hepatocellularcarcinoma, Hurthle cell carcinoma, hyaline carcinoma, hypemephroidcarcinoma, infantile embryonal carcinoma, carcinoma in situ,intraepidermal carcinoma, intraepithelial carcinoma, Krompecher'scarcinoma, Kulchitzky-cell carcinoma, large-cell carcinoma, lenticularcarcinoma, carcinoma lenticulare, lipomatous carcinoma, lymphoepithelialcarcinoma, carcinoma medullare, medullary carcinoma, melanoticcarcinoma, carcinoma molle, mucinous carcinoma, carcinoma muciparum,carcinoma mucocellulare, mucoepidermoid carcinoma, carcinoma mucosum,mucous carcinoma, carcinoma myxomatodes, nasopharyngeal carcinoma, oatcell carcinoma, carcinoma ossificans, osteoid carcinoma, papillarycarcinoma, periportal carcinoma, preinvasive carcinoma, prickle cellcarcinoma, pultaceous carcinoma, renal cell carcinoma of kidney, reservecell carcinoma, carcinoma sarcomatodes, schneiderian carcinoma,scirrhous carcinoma, carcinoma scroti, signet-ring cell carcinoma,carcinoma simplex, small-cell carcinoma, solanoid carcinoma, spheroidalcell carcinoma, spindle cell carcinoma, carcinoma spongiosum, squamouscarcinoma, squamous cell carcinoma, string carcinoma, carcinomatelangiectaticum, carcinoma telangiectodes, transitional cell carcinoma,carcinoma tuberosum, tuberous carcinoma, verrucous carcinoma, andcarcinoma villosum.

“Anti-cancer agent” is used in accordance with its plain and ordinarymeaning and refers to a composition (e.g., compound, drug, antagonist,inhibitor, modulator) having antineoplastic properties or the ability toinhibit the growth or proliferation of cells. In some embodiments, ananti-cancer agent is a chemotherapeutic. An anti-cancer agent may be anagent approved by the FDA or similar regulatory agency of a countryother than the USA, for treating cancer.

Examples of anti-cancer agents include, but are not limited to, MEK(e.g., MEK1, MEK2, or MEK1 and MEK2) inhibitors (e.g., XL518, CI-1040,PD035901, selumetinib/AZD6244, GSK1120212/trametinib, GDC-0973,ARRY-162, ARRY-300, AZD8330, PD0325901, U0126, PD98059, TAK-733,PD318088, AS703026, BAY 869766), alkylating agents (e.g.,cyclophosphamide, ifosfamide, chlorambucil, busulfan, melphalan,mechlorethamine, uramustine, thiotepa, nitrosoureas, nitrogen mustards(e.g., mechloroethamine, cyclophosphamide, chlorambucil, meiphalan),ethylenimine and methylmelamines (e.g., hexamethlymelamine, thiotepa),alkyl sulfonates (e.g., busulfan), nitrosoureas (e.g., carmustine,lomusitne, semustine, streptozocin), triazenes (decarbazine)),anti-metabolites (e.g., 5-azathioprine, leucovorin, capecitabine,fludarabine, gemcitabine, pemetrexed, raltitrexed, folic acid analog(e.g., methotrexate), or pyrimidine analogs (e.g., fluorouracil,floxouridine, Cytarabine), purine analogs (e.g., mercaptopurine,thioguanine, pentostatin), etc.), plant alkaloids (e.g., vincristine,vinblastine, vinorelbine, vindesine, podophyllotoxin, paclitaxel,docetaxel, etc.), topoisomerase inhibitors (e.g., irinotecan, topotecan,amsacrine, etoposide (VP16), etoposide phosphate, teniposide, etc.),antitumor antibiotics (e.g., doxorubicin, adriamycin, daunorubicin,epirubicin, actinomycin, bleomycin, mitomycin, mitoxantrone, plicamycin,etc.), platinum-based compounds (e.g., cisplatin, oxaloplatin,carboplatin), anthracenedione (e.g., mitoxantrone), substituted urea(e.g., hydroxyurea), methyl hydrazine derivative (e.g., procarbazine),adrenocortical suppressant (e.g., mitotane, aminoglutethimide),epipodophyllotoxins (e.g., etoposide), antibiotics (e.g., daunorubicin,doxorubicin, bleomycin), enzymes (e.g., L-asparaginase), inhibitors ofmitogen-activated protein kinase signaling (e.g., U0126, PD98059,PD184352, PD0325901, ARRY-142886, SB239063, SP600125, BAY 43-9006,wortmannin, or LY294002, Syk inhibitors, mTOR inhibitors, antibodies(e.g., rituxan), gossyphol, genasense, polyphenol E, Chlorofusin, alltrans-retinoic acid (ATRA), bryostatin, tumor necrosis factor-relatedapoptosis-inducing ligand (TRAIL), 5-aza-2′-deoxycytidine, all transretinoic acid, doxorubicin, vincristine, etoposide, gemcitabine,imatinib (Gleevec®), geldanamycin,17-N-Allylamino-17-Demethoxygeldanamycin (17-AAG), flavopiridol,LY294002, bortezomib, trastuzumab, BAY 11-7082, PKC412, PD184352,20-epi-1, 25 dihydroxyvitamin D3; 5-ethynyluracil; abiraterone;aclarubicin; acylfulvene; adecypenol; adozelesin; aldesleukin; ALL-TKantagonists; altretamine; ambamustine; amidox; amifostine;aminolevulinic acid; amrubicin; amsacrine; anagrelide; anastrozole;andrographolide; angiogenesis inhibitors; antagonist D; antagonist G;antarelix; anti-dorsalizing morphogenetic protein-1; antiandrogen,prostatic carcinoma; antiestrogen; antineoplaston; antisenseoligonucleotides; aphidicolin glycinate; apoptosis gene modulators;apoptosis regulators; apurinic acid; ara-CDP-DL-PTBA; argininedeaminase; asulacrine; atamestane; atrimustine; axinastatin 1;axinastatin 2; axinastatin 3; azasetron; azatoxin; azatyrosine; baccatinIII derivatives; balanol; batimastat; BCR/ABL antagonists;benzochlorins; benzoylstaurosporine; beta lactam derivatives;beta-alethine; betaclamycin B; betulinic acid; bFGF inhibitor;bicalutamide; bisantrene; bisaziridinylspermine; bisnafide; bistrateneA; bizelesin; breflate; bropirimine; budotitane; buthionine sulfoximine;calcipotriol; calphostin C; camptothecin derivatives; canarypox IL-2;capecitabine; carboxamide-amino-triazole; carboxyamidotriazole; CaRestM3; CARN 700; cartilage derived inhibitor; carzelesin; casein kinaseinhibitors (ICOS); castanospermine; cecropin B; cetrorelix; chlorins;chloroquinoxaline sulfonamide; cicaprost; cis-porphyrin; cladribine;clomifene analogues; clotrimazole; collismycin A; collismycin B;combretastatin A4; combretastatin analogue; conagenin; crambescidin 816;crisnatol; cryptophycin 8; cryptophycin A derivatives; curacin A;cyclopentanthraquinones; cycloplatam; cypemycin; cytarabine ocfosfate;cytolytic factor; cytostatin; dacliximab; decitabine; dehydrodidemnin B;deslorelin; dexamethasone; dexifosfamide; dexrazoxane; dexverapamil;diaziquone; didemnin B; didox; diethylnorspermine;dihydro-5-azacytidine; 9-dioxamycin; diphenyl spiromustine; docosanol;dolasetron; doxifluridine; droloxifene; dronabinol; duocarmycin SA;ebselen; ecomustine; edelfosine; edrecolomab; eflornithine; elemene;emitefur; epirubicin; epristeride; estramustine analogue; estrogenagonists; estrogen antagonists; etanidazole; etoposide phosphate;exemestane; fadrozole; fazarabine; fenretinide; filgrastim; finasteride;flavopiridol; flezelastine; fluasterone; fludarabine; fluorodaunorunicinhydrochloride; forfenimex; formestane; fostriecin; fotemustine;gadolinium texaphyrin; gallium nitrate; galocitabine; ganirelix;gelatinase inhibitors; gemcitabine; glutathione inhibitors; hepsulfam;heregulin; hexamethylene bisacetamide; hypericin; ibandronic acid;idarubicin; idoxifene; idramantone; ilmofosine; ilomastat;imidazoacridones; imiquimod; immunostimulant peptides; insulin-likegrowth factor-1 receptor inhibitor; interferon agonists; interferons;interleukins; iobenguane; iododoxorubicin; ipomeanol, 4-; iroplact;irsogladine; isobengazole; isohomohalicondrin B; itasetron;jasplakinolide; kahalalide F; lamellarin-N triacetate; lanreotide;leinamycin; lenograstim; lentinan sulfate; leptolstatin; letrozole;leukemia inhibiting factor; leukocyte alpha interferon;leuprolide+estrogen+progesterone; leuprorelin; levamisole; liarozole;linear polyamine analogue; lipophilic disaccharide peptide; lipophilicplatinum compounds; lissoclinamide 7; lobaplatin; lombricine;lometrexol; lonidamine; losoxantrone; lovastatin; loxoribine;lurtotecan; lutetium texaphyrin; lysofylline; lytic peptides;maitansine; mannostatin A; marimastat; masoprocol; maspin; matrilysininhibitors; matrix metalloproteinase inhibitors; menogaril; merbarone;meterelin; methioninase; metoclopramide; MIF inhibitor; mifepristone;miltefosine; mirimostim; mismatched double stranded RNA; mitoguazone;mitolactol; mitomycin analogues; mitonafide; mitotoxin fibroblast growthfactor-saporin; mitoxantrone; mofarotene; molgramostim; monoclonalantibody, human chorionic gonadotrophin; monophosphoryl lipidA+myobacterium cell wall sk; mopidamol; multiple drug resistance geneinhibitor; multiple tumor suppressor 1-based therapy; mustard anticanceragent; mycaperoxide B; mycobacterial cell wall extract; myriaporone;N-acetyldinaline; N-substituted benzamides; nafarelin; nagrestip;naloxone+pentazocine; napavin; naphterpin; nartograstim; nedaplatin;nemorubicin; neridronic acid; neutral endopeptidase; nilutamide;nisamycin; nitric oxide modulators; nitroxide antioxidant; nitrullyn;O6-benzylguanine; octreotide; okicenone; oligonucleotides; onapristone;ondansetron; ondansetron; oracin; oral cytokine inducer; ormaplatin;osaterone; oxaliplatin; oxaunomycin; palauamine; palmitoylrhizoxin;pamidronic acid; panaxytriol; panomifene; parabactin; pazelliptine;pegaspargase; peldesine; pentosan polysulfate sodium; pentostatin;pentrozole; perflubron; perfosfamide; perillyl alcohol; phenazinomycin;phenylacetate; phosphatase inhibitors; picibanil; pilocarpinehydrochloride; pirarubicin; piritrexim; placetin A; placetin B;plasminogen activator inhibitor; platinum complex; platinum compounds;platinum-triamine complex; porfimer sodium; porfiromycin; prednisone;propyl bis-acridone; prostaglandin J2; proteasome inhibitors; proteinA-based immune modulator; protein kinase C inhibitor; protein kinase Cinhibitors, microalgal; protein tyrosine phosphatase inhibitors; purinenucleoside phosphorylase inhibitors; purpurins; pyrazoloacridine;pyridoxylated hemoglobin polyoxyethylerie conjugate; raf antagonists;raltitrexed; ramosetron; ras farnesyl protein transferase inhibitors;ras inhibitors; ras-GAP inhibitor; retelliptine demethylated; rhenium Re186 etidronate; rhizoxin; ribozymes; RII retinamide; rogletimide;rohitukine; romurtide; roquinimex; rubiginone B1; ruboxyl; safingol;saintopin; SarCNU; sarcophytol A; sargramostim; Sdi 1 mimetics;semustine; senescence derived inhibitor 1; sense oligonucleotides;signal transduction inhibitors; signal transduction modulators; singlechain antigen-binding protein; sizofuran; sobuzoxane; sodiumborocaptate; sodium phenylacetate; solverol; somatomedin bindingprotein; sonermin; sparfosic acid; spicamycin D; spiromustine;splenopentin; spongistatin 1; squalamine; stem cell inhibitor; stem-celldivision inhibitors; stipiamide; stromelysin inhibitors; sulfinosine;superactive vasoactive intestinal peptide antagonist; suradista;suramin; swainsonine; synthetic glycosaminoglycans; tallimustine;tamoxifen methiodide; tauromustine; tazarotene; tecogalan sodium;tegafur; tellurapyrylium; telomerase inhibitors; temoporfin;temozolomide; teniposide; tetrachlorodecaoxide; tetrazomine;thaliblastine; thiocoraline; thrombopoietin; thrombopoietin mimetic;thymalfasin; thymopoietin receptor agonist; thymotrinan; thyroidstimulating hormone; tin ethyl etiopurpurin; tirapazamine; titanocenebichloride; topsentin; toremifene; totipotent stem cell factor;translation inhibitors; tretinoin; triacetyluridine; triciribine;trimetrexate; triptorelin; tropisetron; turosteride; tyrosine kinaseinhibitors; tyrphostins; UBC inhibitors; ubenimex; urogenitalsinus-derived growth inhibitory factor; urokinase receptor antagonists;vapreotide; variolin B; vector system, erythrocyte gene therapy;velaresol; veramine; verdins; verteporfin; vinorelbine; vinxaltine;vitaxin; vorozole; zanoterone; zeniplatin; zilascorb; zinostatinstimalamer, Adriamycin, Dactinomycin, Bleomycin, Vinblastine, Cisplatin,acivicin; aclarubicin; acodazole hydrochloride; acronine; adozelesin;aldesleukin; altretamine; ambomycin; ametantrone acetate;aminoglutethimide; amsacrine; anastrozole; anthramycin; asparaginase;asperlin; azacitidine; azetepa; azotomycin; batimastat; benzodepa;bicalutamide; bisantrene hydrochloride; bisnafide dimesylate; bizelesin;bleomycin sulfate; brequinar sodium; bropirimine; busulfan;cactinomycin; calusterone; caracemide; carbetimer; carboplatin;carmustine; carubicin hydrochloride; carzelesin; cedefingol;chlorambucil; cirolemycin; cladribine; crisnatol mesylate;cyclophosphamide; cytarabine; dacarbazine; daunorubicin hydrochloride;decitabine; dexormaplatin; dezaguanine; dezaguanine mesylate;diaziquone; doxorubicin; doxorubicin hydrochloride; droloxifene;droloxifene citrate; dromostanolone propionate; duazomycin; edatrexate;eflornithine hydrochloride; elsamitrucin; enloplatin; enpromate;epipropidine; epirubicin hydrochloride; erbulozole; esorubicinhydrochloride; estramustine; estramustine phosphate sodium; etanidazole;etoposide; etoposide phosphate; etoprine; fadrozole hydrochloride;fazarabine; fenretinide; floxuridine; fludarabine phosphate;fluorouracil; fluorocitabine; fosquidone; fostriecin sodium;gemcitabine; gemcitabine hydrochloride; hydroxyurea; idarubicinhydrochloride; ifosfamide; iimofosine; interleukin I1 (includingrecombinant interleukin II, or rlL.sub.2), interferon alfa-2a;interferon alfa-2b; interferon alfa-n1; interferon alfa-n3; interferonbeta-1a; interferon gamma-1b; iproplatin; irinotecan hydrochloride;lanreotide acetate; letrozole; leuprolide acetate; liarozolehydrochloride; lometrexol sodium; lomustine; losoxantrone hydrochloride;masoprocol; maytansine; mechlorethamine hydrochloride; megestrolacetate; melengestrol acetate; melphalan; menogaril; mercaptopurine;methotrexate; methotrexate sodium; metoprine; meturedepa; mitindomide;mitocarcin; mitocromin; mitogillin; mitomalcin; mitomycin; mitosper;mitotane; mitoxantrone hydrochloride; mycophenolic acid; nocodazoie;nogalamycin; ormaplatin; oxisuran; pegaspargase; peliomycin;pentamustine; peplomycin sulfate; perfosfamide; pipobroman; piposulfan;piroxantrone hydrochloride; plicamycin; plomestane; porfimer sodium;porfiromycin; prednimustine; procarbazine hydrochloride; puromycin;puromycin hydrochloride; pyrazofurin; riboprine; rogletimide; safingol;safingol hydrochloride; semustine; simtrazene; sparfosate sodium;sparsomycin; spirogermanium hydrochloride; spiromustine; spiroplatin;streptonigrin; streptozocin; sulofenur; talisomycin; tecogalan sodium;tegafur; teloxantrone hydrochloride; temoporfin; teniposide; teroxirone;testolactone; thiamiprine; thioguanine; thiotepa; tiazofurin;tirapazamine; toremifene citrate; trestolone acetate; triciribinephosphate; trimetrexate; trimetrexate glucuronate; triptorelin;tubulozole hydrochloride; uracil mustard; uredepa; vapreotide;verteporfin; vinblastine sulfate; vincristine sulfate; vindesine;vindesine sulfate; vinepidine sulfate; vinglycinate sulfate;vinleurosine sulfate; vinorelbine tartrate; vinrosidine sulfate;vinzolidine sulfate; vorozole; zeniplatin; zinostatin; zorubicinhydrochloride, agents that arrest cells in the G2-M phases and/ormodulate the formation or stability of microtubules, (e.g., Taxol™ (i.e.paclitaxel), Taxotere™, compounds comprising the taxane skeleton,Erbulozole (i.e. R-55104), Dolastatin 10 (i.e. DLS-10 and NSC-376128),Mivobulin isethionate (i.e. as CI-980), Vincristine, NSC-639829,Discodermolide (i.e. as NVP-XX-A-296), ABT-751 (Abbott, i.e. E-7010),Altorhyrtins (e.g., Altorhyrtin A and Altorhyrtin C), Spongistatins(e.g., Spongistatin 1, Spongistatin 2, Spongistatin 3, Spongistatin 4,Spongistatin 5, Spongistatin 6, Spongistatin 7, Spongistatin 8, andSpongistatin 9), Cemadotin hydrochloride (i.e. LU-103793 andNSC-D-669356), Epothilones (e.g., Epothilone A, Epothilone B, EpothiloneC (i.e. desoxyepothilone A or dEpoA), Epothilone D (i.e. KOS-862, dEpoB,and desoxyepothilone B), Epothilone E, Epothilone F, Epothilone BN-oxide, Epothilone A N-oxide, 16-aza-epothilone B, 21-aminoepothilone B(i.e. BMS-310705), 21-hydroxyepothilone D (i.e. Desoxyepothilone F anddEpoF), 26-fluoroepothilone, Auristatin PE (i.e. NSC-654663), Soblidotin(i.e. TZT-1027), LS-4559-P (Pharmacia, i.e. LS-4577), LS-4578(Pharmacia, i.e. LS-477-P), LS-4477 (Pharmacia), LS-4559 (Pharmacia),RPR-112378 (Aventis), Vincristine sulfate, DZ-3358 (Daiichi), FR-182877(Fujisawa, i.e. WS-9885B), GS-164 (Takeda), GS-198 (Takeda), KAR-2(Hungarian Academy of Sciences), BSF-223651 (BASF, i.e. ILX-651 andLU-223651), SAH-49960 (Lilly/Novartis), SDZ-268970 (Lilly/Novartis),AM-97 (Armad/Kyowa Hakko), AM-132 (Armad), AM-138 (Armad/Kyowa Hakko),IDN-5005 (Indena), Cryptophycin 52 (i.e. LY-355703), AC-7739 (Ajinomoto,i.e. AVE-8063A and CS-39.HCl), AC-7700 (Ajinomoto, i.e. AVE-8062,AVE-8062A, CS-39-L-Ser.HCl, and RPR-258062A), Vitilevuamide, TubulysinA, Canadensol, Centaureidin (i.e. NSC-106969), T-138067 (Tularik, i.e.T-67, TL-138067 and TI-138067), COBRA-1 (Parker Hughes Institute, i.e.DDE-261 and WHI-261), H10 (Kansas State University), H16 (Kansas StateUniversity), Oncocidin A1 (i.e. BTO-956 and DIE), DDE-313 (Parker HughesInstitute), Fijianolide B, Laulimalide, SPA-2 (Parker Hughes Institute),SPA-1 (Parker Hughes Institute, i.e. SPIKET-P), 3-IAABU(Cytoskeleton/Mt. Sinai School of Medicine, i.e. MF-569), Narcosine(also known as NSC-5366), Nascapine, D-24851 (Asta Medica), A-105972(Abbott), Hemiasterlin, 3-BAABU (Cytoskeleton/Mt. Sinai School ofMedicine, i.e. MF-191), TMPN (Arizona State University), Vanadoceneacetylacetonate, T-138026 (Tularik), Monsatrol, lnanocine (i.e.NSC-698666), 3-IAABE (Cytoskeleton/Mt. Sinai School of Medicine),A-204197 (Abbott), T-607 (Tuiarik, i.e. T-900607), RPR-115781 (Aventis),Eleutherobins (such as Desmethyleleutherobin, Desaetyleleutherobin,lsoeleutherobin A, and Z-Eleutherobin), Caribaeoside, Caribaeolin,Halichondrin B, D-64131 (Asta Medica), D-68144 (Asta Medica),Diazonamide A, A-293620 (Abbott), NPI-2350 (Nereus), Taccalonolide A,TUB-245 (Aventis), A-259754 (Abbott), Diozostatin, (−)-Phenylahistin(i.e. NSCL-96F037), D-68838 (Asta Medica), D-68836 (Asta Medica),Myoseverin B, D-43411 (Zentaris, i.e. D-81862), A-289099 (Abbott),A-318315 (Abbott), HTI-286 (i.e. SPA-110, trifluoroacetate salt)(Wyeth), D-82317 (Zentaris), D-82318 (Zentaris), SC-12983 (NCI),Resverastatin phosphate sodium, BPR-OY-007 (National Health ResearchInstitutes), and SSR-250411 (Sanofi)), steroids (e.g., dexamethasone),finasteride, aromatase inhibitors, gonadotropin-releasing hormoneagonists (GnRH) such as goserelin or leuprolide, adrenocorticosteroids(e.g., prednisone), progestins (e.g., hydroxyprogesterone caproate,megestrol acetate, medroxyprogesterone acetate), estrogens (e.g.,diethlystilbestrol, ethinyl estradiol), antiestrogen (e.g., tamoxifen),androgens (e.g., testosterone propionate, fluoxymesterone), antiandrogen(e.g., flutamide), immunostimulants (e.g., Bacillus Calmette-Guerin(BCG), levamisole, interleukin-2, alpha-interferon, etc.), monoclonalantibodies (e.g., anti-CD20, anti-HER2, anti-CD52, anti-HLA-DR, andanti-VEGF monoclonal antibodies), immunotoxins (e.g., anti-CD33monoclonal antibody-calicheamicin conjugate, anti-CD22 monoclonalantibody-pseudomonas exotoxin conjugate, etc.), radioimmunotherapy(e.g., anti-CD20 monoclonal antibody conjugated to ¹¹¹In, ⁹⁰Y, or ¹³¹I,etc.), triptolide, homoharringtonine, dactinomycin, doxorubicin,epirubicin, topotecan, itraconazole, vindesine, cerivastatin,vincristine, deoxyadenosine, sertraline, pitavastatin, irinotecan,clofazimine, 5-nonyloxytryptamine, vemurafenib, dabrafenib, erlotinib,gefitinib, EGFR inhibitors, epidermal growth factor receptor(EGFR)-targeted therapy or therapeutic (e.g., gefitinib (Iressa™),erlotinib (Tarceva™), cetuximab (Erbitux™), lapatinib (Tykerb™),panitumumab (Vectibix™), vandetanib (Caprelsa™), afatinib/BIBW2992,CI-1033/canertinib, neratinib/HKI-272, CP-724714, TAK-285, AST-1306,ARRY334543, ARRY-380, AG-1478, dacomitinib/PF299804, OSI-420/desmethylerlotinib, AZD8931, AEE788, pelitinib/EKB-569, CUDC-101, WZ8040, WZ4002,WZ3146, AG-490, XL⁶⁴⁷, PD153035, BMS-599626), sorafenib, imatinib,sunitinib, dasatinib, or the like.

“Chemotherapeutic” or “chemotherapeutic agent” is used in accordancewith its plain ordinary meaning and refers to a chemical composition orcompound having antineoplastic properties or the ability to inhibit thegrowth or proliferation of cells.

The term “nanocrystal” and the like refer, in the usual and customarysense, to a polycrystalline material having a crystallite size less thana micrometer (e.g., 1-10, 1-20, 1-30, 1-40, 1-50, 1-100, 1-200-, 1-500,or even 1-999 nm). The terms “scintillator nanocrystal,” “nanocrystalphosphor,” “nanophosphor,” “nanocrystal radiation scintillator” and thelike as used herein refer, in the usual and customary sense, to ananocrystal which includes a scintillator. The term “scintillator” andthe like refer, in the usual and customary sense, to a material thatexhibits luminescence when excited by radiation (e.g., ionizingradiation), as known in the art. Thus, a scintillator nanocrystal canrobustly scintillate (e.g., by releasing photons) in response toradiation. The scintillator may be energized to release photons byexposure to ionizing radiation including, e.g., X-rays, particle beamradiation, and the like. Scintillator nanocrystals can be held togetherby covalent or non-covalent forces, as known in the art.

The term “scintillator-activated photocleavable linker” and the likerefer, in the usual and customary sense, to a divalent chemical linkerwhich can undergo cleavage upon exposure to light, e.g., light emittedby a scintillator nanocrystal in response to exposure to radiation.

A “detectable moiety” is a composition detectable by spectroscopic,photochemical, biochemical, immunochemical, chemical, magnetic resonanceimaging, or other physical means. For example, useful detectablemoieties include ³²P, fluorescent dyes, electron-dense reagents, biotin,digoxigenin, paramagnetic molecules, superparamagnetic iron oxide,monocrystalline iron oxide, Gadolinium chelate (“Gd-chelate”) molecules,Gadolinium, radioisotopes, radionuclides (e.g., carbon-11, nitrogen-13,oxygen-15, fluorine-18, rubidium-82), fluorodeoxyglucose (e.g.,fluorine-18 labeled), any gamma ray emitting radionuclides,positron-emitting radionuclide, iodinated contrast agents (e.g.,iohexol, iodixanol, ioversol, iopamidol, ioxilan, iopromide,diatrizoate, metrizoate, ioxaglate), barium sulfate, thorium dioxide,gold, fluorophores, two-photon fluorophores, or radionuclides such as⁴⁷Sc, ⁶⁴Cu, ⁶⁷Cu, ⁸⁹Sr, ⁸⁶Y, ⁸⁷Y, ⁹⁰Y, ¹⁰⁵Rh, ¹¹¹Ag, ¹¹¹In, ^(117m)Sn,¹⁴⁹Pm, ¹⁵³Sm, ¹⁶⁶Ho, ¹⁷⁷Lu, ¹⁸⁶Re, ¹⁸⁸Re, ²¹¹At, or ²¹²Bi.

The terms “YAG-Pr” and “YAG-Pr³⁺” refer, in the usual and customarysense, to yttrium aluminum garnet doped with praseodymium (e.g.,Y₃Al₅O₁₂:Pr).

Absent express indication otherwise, the term “about” in the context ofa numeric value refers to the nominal numeric value +/−10% thereof.

Compositions

In a first aspect, there is provided a composition including ascintillator nanocrystal linked to a chemical agent moiety through ascintillator-activated photocleavable linker.

Further to any composition disclosed herein, the scintillatornanocrystal can have a diameter (e.g., the longest dimension or thenanocrystal) from about 25 nm to about 300 nm, e.g., about 25-300 nm,about 25-250 nm, about 25-200, about 25-150 nm, about 25-100 nm, about25-75 nm or about 25-50 nm. The scintillator nanocrystal can havediameter of about 25 nm, 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 50 nm, 70nm, 80 nm, 90 nm, 100 nm, 125 nm, 150 nm, 175 nm, 200 nm, 250 nm or 300nm. The scintillator nanocrystal can have diameter from about 50 nm toabout 250 nm, about 50 nm to about 200 nm, about 50 nm or about 150 nm,or about 50 nm to about 100 nm. The longest dimension of thescintillator nanocrystal can be about 1000 nm or less, or about 100 nmor less, or about 10 nm or less. The scintillator nanocrystal can have alongest dimension of about 10-1000 nm, about 10-500 nm, about 10-400 nm,about 10-300 nm, about 10-200 nm, or about 10-100 nm. The longestdimension can be about 5 nm, 10 nm, 20 nm, 30 nm, 40 nm, 50 nm, 60 nm,70 nm, 80 nm, 90 nm, 100 nm, 110 nm, 120 nm, 130 nm, 140 nm, 150 nm, 160nm, 170 nm, 180 nm, 190 nm, 200 nm, 220 nm, 240 nm, 260 nm, 280 nm, 300nm, 350 nm, 400 nm, 500 nm or even 999 nm.

The term “chemical agent moiety” and the like in the context ofcompositions and methods disclosed herein refer to a monovalent compoundentity attached to the remainder of a composition described herein. Inembodiments, the chemical agent moiety can elicit a chemical orbiochemical reaction when released from the scintillator nanocrystalthrough cleavage of the scintillator-activated photocleavable linker.Thus, the methods and compositions disclosed herein may be useful, forexample, for controlled delivery of chemical agent moieties via specifictemporal and spatial activation. For example, in medical applications,the underlying concept may be to restrict toxic drug effects to tumorsand infected tissue by delivering nontoxic, inactive nanoscintillatorlinked drug selectively to the tumor. A radiation beam (e.g., a low doseradiation beam) can activate the scintillator nanocrystal component,which in turn can emit light to break the scintillator-activatedphotocleavable linker, resulting in a chemical agent moiety (e.g., drug)being released in an active form at the site of the radiation exposure.Indeed, this can be very localized or include the whole body. Thecompositions and methods disclosed herein can be useful for tissueengineering, release of hormones or antibiotics, and industrialapplications in manufacturing and repair of pipes and coverings withrelease of multiple components for cements and other agents in a timeand location controlled manner in electronics, microfabrication, optics,and related fields.

In embodiments, the chemical agent moiety can be a prodrug. The terms“prodrug,” “prodrug moiety” and the like in reference to an embodimentof a compound described herein, refers to a compound that can undergocleavage from the scintillator nanocrystal to provide a chemical moietysuch as a physiologically active agent. Cleavage can be affected bycleavage of a scintillator-activated photocleavage linker, resulting inrelease of the chemical agent moiety (e.g., a physiologically activeagent, such as a drug).

The scintillator nanocrystal can include a plurality of scintillatoractivators dispersed within a host crystal lattice. The terms“activator” in the context of scintillators, “scintillator activator”and the like refer to a chemical species added (e.g., as a dopant) to acrystal (e.g., a nanocrystal) capable of facilitating luminescence(e.g., in response to radiation). In embodiments, the scintillatoractivator exhibits a high quantum efficiency when excited by radiationto emit photons. The scintillator activator can be any one or more ofCu, Ag, Eu, Ce, Th, Ce, or Pr. The scintillator activator can be any oneor more of Ce, Eu or Pr. The scintillator activator can be Ce, Eu or Pr.The scintillator activators can be Ce or Pr. The scintillator activatorcan be Ce. The scintillator activators can be Eu. The scintillatoractivators can be Pr. Typically, the scintillator nanocrystal includes aplurality of scintillator activators. The plurality of scintillatoractivators may be spaced to minimize concentration quenching. Theplurality of scintillator activators may be spaced to maximize theoverall photon release, luminescence or scintillation from thescintillator nanocrystal to the scintillator-activated photocleavablelinker. In embodiments, where the scintillator activator is Pr, theplurality of Pr scintillator activators may be spaced at approximatelythe Pr—O bond length.

In embodiments, the plurality of scintillator activators constituteabout 1 atomic percent of the scintillator nanocrystal. In embodiments,the plurality of scintillator activators constitute about 2 atomicpercent of the scintillator nanocrystal. In embodiments, the pluralityof scintillator activators constitute about 3 atomic percent of thescintillator nanocrystal. In embodiments, the plurality of scintillatoractivators constitute about 4 atomic percent of the scintillatornanocrystal. In embodiments, the plurality of scintillator activatorsconstitute about 5 atomic percent of the scintillator nanocrystal. Inembodiments, the plurality of scintillator activators constitute about 6atomic percent of the scintillator nanocrystal. In embodiments, theplurality of scintillator activators constitute about 7 atomic percentof the scintillator nanocrystal. In embodiments, the plurality ofscintillator activators constitute about 8 atomic percent of thescintillator nanocrystal. In embodiments, the plurality of scintillatoractivators constitute about 9 atomic percent of the scintillatornanocrystal. In embodiments, the plurality of scintillator activatorsconstitute about 10 atomic percent of the scintillator nanocrystal. Inembodiments, the plurality of scintillator activators constitute about1-10 atomic percent of the scintillator nanocrystal. In embodiments, theplurality of scintillator activators constitute about 2-9 atomic percentof the scintillator nanocrystal. In embodiments, the plurality ofscintillator activators constitute about 3-8 atomic percent of thescintillator nanocrystal. In embodiments, the plurality of scintillatoractivators constitute about 4-6 atomic percent of the scintillatornanocrystal.

In embodiments, the host crystal lattice is composed of biocompatiblematerials (e.g., materials that are biologically inert or do notsubstantially interfere with organism (e.g., human biological function).Further to any composition disclosed herein, the host crystal latticecan be a chloride host crystal lattice, bromide host crystal lattice,oxide host crystal lattice, iodide host crystal lattice or silicate hostcrystal lattice. The term “host crystal lattice” and the like refer, inthe usual and customary sense, to a plurality of chemical atoms boundtogether in an approximate lattice structure comprising a plurality ofcavities in which molecules of a second, guest component (e.g., ascintillator activator) can reside. Thus, the guest component can be anactivator, e.g., an activator as disclosed herein. A “chloride hostcrystal lattice” is a host crystal lattice which includes chlorineatoms. A “bromide host crystal lattice” is a host crystal lattice whichincludes bromine atoms. An “oxide host crystal lattice” is a hostcrystal lattice which includes oxygen atoms. An “iodide host crystallattice” is a host crystal lattice which includes iodine atoms. A“silicate host crystal lattice” is a host crystal lattice which includesa silicate, as known in the art. The host crystal lattice can be alanthium bromide host crystal lattice, as known in the art, whichincludes lanthanum and bromine. The host crystal lattice can be an oxidehost crystal lattice or silicate host crystal lattice. The host crystallattice can be an oxide host crystal lattice. The host crystal latticecan be a silicate host crystal lattice.

Further to any composition disclosed herein, the host crystal latticecan be a garnet host crystal lattice. The term “garnet host crystallattice” and the like refer, in the usual and customary sense, to acrystalline compound as known in the art including a transition metal orlanthanoid, e.g., Lu, Yb, Tm, Er, Y, Ho, Dy or Tb. The garnet hostcrystal lattice can include aluminum. The host crystal lattice can be anyttrium aluminum oxide (YAG, e.g., Y₃Al₅O₁₂) host crystal lattice, asknown in the art. The host crystal lattice can be a gadolinium/yttriumaluminum oxide host crystal lattice or yttrium gallium/aluminum oxidehost crystal lattice, as known in the art. The host crystal lattice canbe an gadolinium/yttrium aluminum oxide host crystal lattice. The hostcrystal lattice can be a yttrium gallium/aluminum oxide host crystallattice.

The scintillator nanocrystal can have the formula(Y_(1-x)Pr_(x))₃Al₅O₁₂, wherein x is 0.0075, 0.01, 0.0125, 0.015 or0.0175.

In embodiments, the scintillator nanocrystal emits a photons afterexcitation from radiation at a wavelength sufficient to cleave thephotocleavable linker without damaging biological structures such ascells, animal organs or animal tissue. Further to any compositiondisclosed herein, the scintillator nanocrystal can emit a photon withemission peaks within (from) about 300 nm to 470 nm. The scintillatornanocrystal can emit a photon with emission peak within (from) about 350nm to 470 nm. The scintillator nanocrystal can emit a photon withemission peak within (from) about 350 nm to 400 nm. The scintillatornanocrystal can emit a photon with emission peak within (from) about 350nm to 370 nm. The scintillator nanocrystal can emit a photon withemission peak within (from) about 350 nm to 360 nm. The scintillatornanocrystal can emit a photon with emission peak within (from) about 100nm to 600 nm.

Further to any composition disclosed herein, the scintillator-activatedphotocleavable linker can be covalently attached to the chemical agentmoiety and a surface of the scintillator nanocrystal. The surface of thescintillator nanocrystal can be a lipid bilayer surface or a silinatedsurface. The surface can be a lipid bilayer surface, as known in theart. The surface can be a silinated surface, as known in the art.

Further to any composition disclosed herein, the scintillator-activatedphotocleavable linker (also referred to herein as the “photocleavablelinker”) can have the formula:

wherein, L¹ and L² are independently bond, —C(O)—, —C(O)O—, —O—, —S—,—NH—, —C(O)NH—, —NHC(O)—, —S(O)₂—, —S(O)NH—, substituted orunsubstituted alkylene, substituted or unsubstituted heteroalkylene,substituted or unsubstituted cycloalkylene, substituted or unsubstitutedheterocycloalkylene, substituted or unsubstituted arylene, orsubstituted or unsubstituted heteroarylene; R¹ is independently halogen,—N₃, —CF₃, —CCl₃, —CBr₃, —CI₃, —CN, —COR², —OR², —NR²R³, —C(O)OR²,—C(O)NR²R³, —NO₂, —SR², —S(O)_(n1)R², —S(O)_(n1)OR², —S(O)_(n1)NR²R³,—NHNR²R³, —ONR²R³, —NHC(O)NHNR²R³, substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heterocycloalkyl, substitutedor unsubstituted aryl, or substituted or unsubstituted heteroaryl; R²and R³ are independently hydrogen, halogen, —N₃, —CF₃, —CCl₃, —CBr₃,—CI₃, —CN, —COH, —OH, —NH₂, —C(O)OH, —C(O)NH₂, —NO₂, —SH, —S(O)_(n1)H,—S(O)_(n2)OH, —S(O)_(n2)NH₂, —NHNH₂, —ONH₂, —NHC(O)NHNH₂, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, or substituted orunsubstituted heteroaryl; n is an integer from 0 to 4; n1 and n2 areindependently 1 or 2.

R¹ can be halogen. R¹ can be —N₃. R¹ can be —CF₃. R¹ can be —CCl₃. R¹can be —CBr₃. R¹ can be —CI₃. R¹ can be —CN. R¹ can be —COR². R¹ can be—OR². R¹ can be —NR²R³. R¹ can be —C(O)OR². R¹ can be —C(O)NR²R³. R¹ canbe —NO₂. R¹ can be —SR². R¹ can be —S(O)_(n1)R². R¹ can be—S(O)_(n1)OR². R¹ can be —S(O)_(n1)NR²R³. R¹ can be —NHNR²R³. R¹ can be—ONR²R³. R¹ can be —NHC(O)NHNR²R³. R¹ can be substituted orunsubstituted alkyl. R¹ can be substituted or unsubstituted heteroalkyl.R¹ can be substituted or unsubstituted cycloalkyl. R¹ can be substitutedor unsubstituted heterocycloalkyl. R¹ can be substituted orunsubstituted aryl. R¹ can be or substituted or unsubstitutedheteroaryl.

In embodiments, R¹ is independently halogen, —N₃, —CF₃, —CCl₃, —CBr₃,—CI₃, —CN, —COR², —OR², —NR²R³, —C(O)OR², —C(O)NR²R³, —NO₂, —SR²,—S(O)_(n1)R², —S(O)_(n1)OR², —S(O)_(n1)NR²R³, —NHNR²R³, —ONR²R³,—NHC(O)NHNR²R³, R^(1A)-substituted or unsubstituted alkyl,R^(1A)-substituted or unsubstituted heteroalkyl, R^(1A)-substituted orunsubstituted cycloalkyl, R^(1A)-substituted or unsubstitutedheterocycloalkyl, R^(1A)-substituted or unsubstituted aryl, orR^(1A)-substituted or unsubstituted heteroaryl. In embodiments, R¹ is—NO₂. In embodiments, R¹ is independently halogen, —N₃, —CF₃, —CCl₃,—CBr₃, —CI₃, —CN, —COR², —OR², —NR²R³, —C(O)OR², —C(O)NR²R³, —NO₂, —SR²,—S(O)_(n1)R², —S(O)_(n1)OR², —S(O)_(n1)NR²R³, —NHNR²R³, —ONR²R³,—NHC(O)NHNR²R³, unsubstituted alkyl, unsubstituted heteroalkyl,unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstitutedaryl, or unsubstituted heteroaryl. In embodiments, R¹ is independentlyhalogen, —N₃, —CF₃, —CCl₃, —CBr₃, —CI₃, —CN, —COR², —OR², —NR²R³,—C(O)OR², —C(O)NR²R³, —NO₂, —SR², —S(O)_(n1)R², —S(O)_(n1)OR²,—S(O)_(n1)NR²R³, —NHNR²R³, —ONR²R³, —NHC(O)NHNR²R³, R^(1A)-substitutedalkyl, R^(1A)-substituted heteroalkyl, R^(1A)-substituted cycloalkyl,R^(1A)-substituted heterocycloalkyl, R^(1A)-substituted aryl, orR^(1A)-substituted heteroaryl. R^(1A) is independently halogen, —N₃,—CF₃, —CCl₃, —CBr₃, —CI₃, —CN, —COR², —OR², —NR²R³, —C(O)OR²,—C(O)NR²R³, —NO₂, —SR², —S(O)_(n1)R², —S(O)_(n1)OR², —S(O)_(n1)NR²R³,—NHNR²R³, —ONR²R³, —NHC(O)NHNR²R³, R^(1B)-substituted or unsubstitutedalkyl, R^(1B)-substituted or unsubstituted heteroalkyl,R^(1B)-substituted or unsubstituted cycloalkyl, R^(1B)-substituted orunsubstituted heterocycloalkyl, R^(1B)-substituted or unsubstitutedaryl, or R^(1B)-substituted or unsubstituted heteroaryl. R1B isindependently halogen, —N₃, —CF₃, —CCl₃, —CBr₃, —CI₃, —CN, —COR², —OR²,—NR²R³, —C(O)OR², —C(O)NR²R³, —NO₂, —SR², —S(O)_(n1)R², —S(O)_(n1)OR²,—S(O)_(n1)NR²R³, —NHNR²R³, —ONR²R³, —NHC(O)NHNR²R³, R^(1C)-substitutedor unsubstituted alkyl, R^(1C)-substituted or unsubstituted heteroalkyl,R^(1C)-substituted or unsubstituted cycloalkyl, R^(1C)-substituted orunsubstituted heterocycloalkyl, R^(1C)-substituted or unsubstitutedaryl, or R^(1C)-substituted or unsubstituted heteroaryl. R^(1C) isindependently halogen, —N₃, —CF₃, —CCl₃, —CBr₃, —CI₃, —CN, —COR², —OR²,—NR²R³, —C(O)OR², —C(O)NR²R³, —NO₂, —SR², —S(O)_(n1)R², —S(O)_(n1)OR²,—S(O)_(n1)NR²R³, —NHNR²R³, —ONR²R³, —NHC(O)NHNR²R³, unsubstituted alkyl,unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstitutedheterocycloalkyl, unsubstituted aryl, or unsubstituted heteroaryl.

Further to any composition disclosed herein including the structure ofFormula (I) and embodiments including, e.g., Formulae (Ia), L¹ or L² canbe a bond. L¹ or L² can be —C(O)—. L¹ or L² can be —C(O)O—. L¹ or L² canbe —O—. L¹ or L² can be —S—. L¹ or L² can be —NH—. L¹ or L² can be—C(O)NH—. L¹ or L² can be —NHC(O)—. L¹ or L² can be —S(O)₂—. L¹ or L²can be —S(O)NH—. L¹ or L² can be substituted or unsubstituted alkylene.L¹ or L² can be substituted or unsubstituted heteroalkylene. L¹ or L²can be substituted or unsubstituted cycloalkylene. L¹ or L² can besubstituted or unsubstituted heterocycloalkylene. L¹ or L² can besubstituted or unsubstituted arylene. L¹ or L² can be or substituted orunsubstituted heteroarylene.

In embodiments, L¹ is a bond, —C(O)—, —C(O)O—, —O—, —S—, —NH—, —C(O)NH—,—NHC(O)—, —S(O)₂—, —S(O)NH—, R^(4A)-substituted or unsubstitutedalkylene, R^(4A)-substituted or unsubstituted heteroalkylene,R^(4A)-substituted or unsubstituted cycloalkylene, R^(4A)-substituted orunsubstituted heterocycloalkylene, R^(4A)-substituted or unsubstitutedarylene, or R^(4A)-substituted or unsubstituted heteroarylene. Inembodiments, L¹ is unsubstituted alkylene, unsubstituted heteroalkylene,unsubstituted cycloalkylene, unsubstituted heterocycloalkylene,unsubstituted arylene, or unsubstituted heteroarylene. In embodiments,L¹ is R^(4A)-substituted alkylene, R^(4A)-substituted heteroalkylene,R^(4A)-substituted cycloalkylene, R^(4A)-heterocycloalkylene,R^(4A)-substituted arylene, or R^(4A)-substituted heteroarylene. R^(4A)is halogen, —CF₃, —CN, —OH, —NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₂Cl,—SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂, —NHC═(O)NHNH₂, —NHC═(O) NH₂,—NHSO₂H, —NHC═(O)H, —NHC(O)—OH, —NHOH, —OCF₃, —OCHF₂, R^(4B)-substitutedor unsubstituted alkyl, R^(4B)-substituted or unsubstituted heteroalkyl,R^(4B)-substituted or unsubstituted cycloalkyl, R^(4B)-substituted orunsubstituted heterocycloalkyl, R^(4B)-substituted or unsubstitutedaryl, or R^(4B)-substituted or unsubstituted heteroaryl. R^(4B) ishalogen, —CF₃, —CN, —OH, —NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₂Cl, —SO₃H,—SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂, —NHC═(O)NHNH₂, —NHC═(O) NH₂, —NHSO₂H,—NHC═(O)H, —NHC(O)—OH, —NHOH, —OCF₃, —OCHF₂, R^(4C)-substituted orunsubstituted alkyl, R^(4C)-substituted or unsubstituted heteroalkyl,R^(4C)-substituted or unsubstituted cycloalkyl, R^(4C)-substituted orunsubstituted heterocycloalkyl, R^(4C)-substituted or unsubstitutedaryl, or R^(4C)-substituted or unsubstituted heteroaryl. R^(4C) ishalogen, —CF₃, —CN, —OH, —NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₂Cl, —SO₃H,—SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂, —NHC═(O)NHNH₂, —NHC═(O)NH₂, —NHSO₂H,—NHC═(O)H, —NHC(O)—OH, —NHOH, —OCF₃, —OCHF₂, unsubstituted alkyl,unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstitutedheterocycloalkyl, unsubstituted aryl, or unsubstituted heteroaryl.

In embodiments, L² is a bond, —C(O)—, —C(O)O—, —O—, —S—, —NH—, —C(O)NH—,—NHC(O)—, —S(O)₂—, —S(O)NH—, R^(5A)-substituted or unsubstitutedalkylene, R^(5A)-substituted or unsubstituted heteroalkylene,R^(5A)-substituted or unsubstituted cycloalkylene, R^(5A)-substituted orunsubstituted heterocycloalkylene, R^(5A)-substituted or unsubstitutedarylene, or R^(5A)-substituted or unsubstituted heteroarylene. Inembodiments, L² is unsubstituted alkylene, unsubstituted heteroalkylene,unsubstituted cycloalkylene, unsubstituted heterocycloalkylene,unsubstituted arylene, or unsubstituted heteroarylene. In embodiments,L² is R^(5A)-substituted alkylene, R^(5A)-substituted heteroalkylene,R^(5A)-substituted cycloalkylene, R^(5A)-heterocycloalkylene,R^(5A)-substituted arylene, or R^(5A)-substituted heteroarylene. R^(5A)is halogen, —CF₃, —CN, —OH, —NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₂Cl,—SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂, —NHC═(O)NHNH₂, —NHC═(O) NH₂,—NHSO₂H, —NHC═(O)H, —NHC(O)—OH, —NHOH, —OCF₃, —OCHF₂, R^(5B)-substitutedor unsubstituted alkyl, R^(5B)-substituted or unsubstituted heteroalkyl,R^(5B)-substituted or unsubstituted cycloalkyl, R^(5B)-substituted orunsubstituted heterocycloalkyl, R^(5B)-substituted or unsubstitutedaryl, or R^(5B)-substituted or unsubstituted heteroaryl. R^(5B) ishalogen, —CF₃, —CN, —OH, —NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₂Cl, —SO₃H,—SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂, —NHC═(O)NHNH₂, —NHC═(O) NH₂, —NHSO₂H,—NHC═(O)H, —NHC(O)—OH, —NHOH, —OCF₃, —OCHF₂, R^(5C)-substituted orunsubstituted alkyl, R^(5C)-substituted or unsubstituted heteroalkyl,R^(5C)-substituted or unsubstituted cycloalkyl, R^(5C)-substituted orunsubstituted heterocycloalkyl, R^(5C)-substituted or unsubstitutedaryl, or R^(5C)-substituted or unsubstituted heteroaryl. R^(5C) ishalogen, —CF₃, —CN, —OH, —NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₂Cl, —SO₃H,—SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂, —NHC═(O)NHNH₂, —NHC═(O) NH₂, —NHSO₂H,—NHC═(O)H, —NHC(O)—OH, —NHOH, —OCF₃, —OCHF₂, unsubstituted alkyl,unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstitutedheterocycloalkyl, unsubstituted aryl, or unsubstituted heteroaryl.

In embodiments, R² or R³ can be hydrogen. R² or R³ can be halogen. R² orR³ can be —N₃. R² or R³ can be —CF₃. R² or R³ can be —CCl₃. R² or R³ canbe —CBr₃. R² or R³ can be —CI₃. R² or R³ can be —CN. R² or R³ can be—COH. R² or R³ can be —OH. R² or R³ can be —NH₂. R² or R³ can be—C(O)OH. R² or R³ can be —C(O)NH₂. R² or R³ can be —NO₂. R² or R³ can be—SH. R² or R³ can be —S(O)_(n1)H. R² or R³ can be —S(O)_(n2)OH. R² or R³can be —S(O)_(n2)NH₂. R² or R³ can be —NHNH₂. R² or R³ can be —ONH₂. R²or R³ can be —NHC(O)NHNH₂. R² or R³ can be substituted or unsubstitutedalkyl. R² or R³ can be substituted or unsubstituted heteroalkyl. R² orR³ can be substituted or unsubstituted cycloalkyl. R² or R³ can besubstituted or unsubstituted heterocycloalkyl. R² or R³ can besubstituted or unsubstituted aryl. R² or R³ can be or substituted orunsubstituted heteroaryl. n can be is an integer from 0 to 4, e.g., 9,1, 2, 3 or 4. n1 and n2 are independently 1 or 2, e.g., 1 or 2.

In embodiments, R² is independently hydrogen, halogen, —N₃, —CF₃, —CCl₃,—CBr₃, —C₃, —CN, —COH, —OH, —NH₂, —C(O)OH, —C(O)NH₂, —NO₂, —SH,—S(O)_(n1)H, —S(O)_(n2)H, —S(O)_(n2)NH₂, —NHNH₂, —ONH₂, —NHC(O)NHNH₂,R^(2A)-substituted or unsubstituted alkyl, R^(2A)-substituted orunsubstituted heteroalkyl, R^(2A)-substituted or unsubstitutedcycloalkyl, R^(2A)-substituted or unsubstituted heterocycloalkyl,R^(2A)-substituted or unsubstituted aryl, or R^(2A)-substituted orunsubstituted heteroaryl, wherein n is an integer from 0 to 4. and n1and n2 are independently 1 or 2. In embodiments, R² is independentlyhydrogen, halogen, —N₃, —CF₃, —CCl₃, —CBr₃, —C₃, —CN, —COH, —OH, —NH₂,—C(O)OH, —C(O)NH₂, —NO₂, —SH, —S(O)_(n1)H, —S(O)_(n2)OH, —S(O)_(n2)NH₂,—NHNH₂, —ONH₂, —NHC(O)NHNH₂, unsubstituted alkyl, unsubstitutedheteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl,unsubstituted aryl, or unsubstituted heteroaryl, wherein n is an integerfrom 0 to 4. and n1 and n2 are independently 1 or 2. In embodiments, R²is independently hydrogen, halogen, —N₃, —CF₃, —CCl₃, —CBr₃, —C₃, —CN,—COH, —OH, —NH₂, —C(O)OH, —C(O)NH₂, substituted alkyl,R^(2A)-substituted heteroalkyl, R^(2A)-substituted cycloalkyl,R^(2A)-substituted heterocycloalkyl, R^(2A)-substituted aryl, orR^(2A)-substituted heteroaryl, wherein n is an integer from 0 to 4. andn1 and n2 are independently 1 or 2. R^(2A) is halogen, —N₃, —CF₃, —CCl₃,—CBr₃, —CI₃, —CN, —COH, —OH, —NH₂, —C(O)OH, —C(O)NH₂, —NO₂, —SH,—S(O)_(n1)H, —S(O)_(n2)OH, —S(O)_(n2)NH₂, —NHNH₂, —ONH₂, —NHC(O)NHNH₂,R^(2B)-substituted or unsubstituted alkyl, R^(2B)-substituted orunsubstituted heteroalkyl, R^(2B)-substituted or unsubstitutedcycloalkyl, R^(2B)-substituted or unsubstituted heterocycloalkyl,R^(2B)-substituted or unsubstituted aryl, or R^(2B)-substituted orunsubstituted heteroaryl, wherein n is an integer from 0 to 4. and n1and n2 are independently 1 or 2. R^(2B) is halogen, —N₃, —CF₃, —CCl₃,—CBr₃, —CI₃, —CN, —COH, —OH, —NH₂, —C(O)OH, —C(O)NH₂, —NO₂, —SH,—S(O)_(n1)H, —S(O)_(n2)OH, —S(O)_(n2)NH₂, —NHNH₂, —ONH₂, —NHC(O)NHNH₂,R^(2C)-substituted or unsubstituted alkyl, R^(2C)-substituted orunsubstituted heteroalkyl, R^(2C)-substituted or unsubstitutedcycloalkyl, R^(2C)-substituted or unsubstituted heterocycloalkyl,R^(2C)-substituted or unsubstituted aryl, or R^(2C)-substituted orunsubstituted heteroaryl, wherein n is an integer from 0 to 4. and n1and n2 are independently 1 or 2. R^(2C) is halogen, —N₃, —CF₃, —CCl₃,—CBr₃, —CI₃, —CN, —COH, —OH, —NH₂, —C(O)OH, —C(O)NH₂, —NO₂, —SH,—S(O)_(n1)H, —S(O)_(n2)OH, —S(O)_(n2)NH₂, —NHNH₂, —ONH₂, —NHC(O)NHNH₂,unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstitutedheterocycloalkyl, unsubstituted aryl, or unsubstituted heteroaryl,wherein n is an integer from 0 to 4. and n1 and n2 are independently 1or 2.

In embodiments, R³ is independently hydrogen, halogen, —N₃, —CF₃, —CCl₃,—CBr₃, —CI₃, —CN, —COH, —OH, —NH₂, —C(O)OH, —C(O)NH₂, —NO₂, —SH,—S(O)_(n1)H, —S(O)_(n2)OH, —S(O)_(n2)NH₂, —NHNH₂, —ONH₂, —NHC(O)NHNH₂,R^(3A)-substituted or unsubstituted alkyl, R^(3A)-substituted orunsubstituted heteroalkyl, R^(3A)-substituted or unsubstitutedcycloalkyl, R^(3A)-substituted or unsubstituted heterocycloalkyl,R^(3A)-substituted or unsubstituted aryl, or R^(3A)-substituted orunsubstituted heteroaryl, wherein n is an integer from 0 to 4. and n1and n2 are independently 1 or 2. In embodiments, R³ is independentlyhydrogen, halogen, —N₃, —CF₃, —CCl₃, —CBr₃, —CI₃, —CN, —COH, —OH, —NH₂,—C(O)OH, —C(O)NH₂, —NO₂, —SH, —S(O)_(n1)H, —S(O)_(n2)OH, —S(O)_(n2)NH₂,—NHNH₂, —ONH₂, —NHC(O)NHNH₂, unsubstituted unsubstituted heteroalkyl,unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstitutedaryl, or unsubstituted heteroaryl, wherein n is an integer from 0 to 4.and n1 and n2 are independently 1 or 2. In embodiments, R³ isindependently hydrogen, halogen, —N₃, —CF₃, —CCl₃, —CBr₃, —CI₃, —CN,—COH, —OH, —NH₂, —C(O)OH, —C(O)NH₂, —NO₂, —SH, —S(O)_(n1)H,—S(O)_(n2)OH, —S(O)_(n2)NH₂, —NHNH₂, —ONH₂, —NHC(O)NHNH₂,R^(3A)-substituted alkyl, R^(3A)-substituted heteroalkyl,R^(3A)-substituted cycloalkyl, R^(3A)-substituted heterocycloalkyl,R^(3A)-substituted aryl, or R^(3A)-substituted heteroaryl, wherein n isan integer from 0 to 4. and n1 and n2 are independently 1 or 2. R^(3A)is halogen, —N₃, —CF₃, —CCl₃, —CBr₃, —CI₃, —CN, —COH, —OH, —NH₂,—C(O)OH, —C(O)NH₂, —NO₂, —SH, —S(O)_(n1)H, —S(O)_(n2)OH, —S(O)_(n2)NH₂,—NHNH₂, —ONH₂, —NHC(O)NHNH₂, R^(3B)-substituted or unsubstituted alkyl,R^(3B)-substituted or unsubstituted heteroalkyl, R^(3B)-substituted orunsubstituted cycloalkyl, R^(3B)-substituted or unsubstitutedheterocycloalkyl, R^(3B)-substituted or unsubstituted aryl, orR^(3B)-substituted or unsubstituted heteroaryl, wherein n is an integerfrom 0 to 4. and n1 and n2 are independently 1 or 2. R^(3B) is halogen,—N₃, —CF₃, —CCl₃, —CBr₃, —CI₃, —CN, —COH, —OH, —NH₂, —C(O)OH, —C(O)NH₂,—NO₂, —SH, —S(O)_(n1)H, —S(O)_(n2)OH, —S(O)_(n2)NH₂, —NHNH₂, —ONH₂,—NHC(O)NHNH₂, R^(3C)-substituted or unsubstituted alkyl,R^(3C)-substituted or unsubstituted heteroalkyl, R^(3C)-substituted orunsubstituted cycloalkyl, R^(3C)-substituted or unsubstitutedheterocycloalkyl, R^(3C)-substituted or unsubstituted aryl, orR^(3C)-substituted or unsubstituted heteroaryl, wherein n is an integerfrom 0 to 4. and n1 and n2 are independently 1 or 2. R^(3C) is halogen,—N₃, —CF₃, —CCl₃, —CBr₃, —CI₃, —CN, —COH, —OH, —NH₂, —C(O)OH, —C(O)NH₂,—NO₂, —SH, —S(O)_(n1)H, —S(O)_(n2)OH, —S(O)_(n2)NH₂, —NHNH₂, —ONH₂,—NHC(O)NHNH₂, unsubstituted heteroalkyl, unsubstituted cycloalkyl,unsubstituted heterocycloalkyl, unsubstituted aryl, or unsubstitutedheteroaryl, wherein n is an integer from 0 to 4. and n1 and n2 areindependently 1 or 2.

Further to any compound and embodiment thereof disclosed herein, inembodiments one or more of R¹, R^(1A), R^(1B), R^(1C), R², R^(2A),R^(2B), R^(2C), R³, R^(3A), R^(3B), R^(3C), R^(4A), R^(4B), R^(4C),R^(5A), R^(5B), R^(5C) is a size-limited substituent, wherein eachsubstituted or unsubstituted alkyl is independently a substituted orunsubstituted C₁-C₂₀ alkyl, each substituted or unsubstitutedheteroalkyl is a substituted or unsubstituted 2 to 20 memberedheteroalkyl, each substituted or unsubstituted cycloalkyl is asubstituted or unsubstituted C₃-C₈ cycloalkyl, and each substituted orunsubstituted heterocycloalkyl is a substituted or unsubstituted 3 to 8membered heterocycloalkyl. Further to any compound and embodimentthereof disclosed herein, in embodiments one or more of R¹, R^(1A),R^(1B), R^(1C), R², R^(2A), R^(2B), R^(2C), R³, R^(3A), R^(3B), R^(3C),R^(4A), R^(4B), R^(4C), R^(5A), R^(5B), R^(5C) is independently a lowersubstituent, wherein each substituted or unsubstituted alkyl is asubstituted or unsubstituted C₁-C₈ alkyl, each substituted orunsubstituted heteroalkyl is a substituted or unsubstituted 2 to 8membered heteroalkyl, each substituted or unsubstituted cycloalkyl is asubstituted or unsubstituted C₃-C₇ cycloalkyl, and each substituted orunsubstituted heterocycloalkyl is a substituted or unsubstituted 3 to 7membered heterocycloalkyl. Further to any compound and embodimentthereof disclosed herein, in embodiments L¹ and/or L² is a size-limitedsubstituent, wherein each substituted or unsubstituted alkyl is asubstituted or unsubstituted C₁-C₂₀ alkylene, each substituted orunsubstituted heteroalkylene is a substituted or unsubstituted 2 to 20membered heteroalkylene, each substituted or unsubstituted cycloalkyleneis a substituted or unsubstituted C₃-C₈ cycloalkylene, and eachsubstituted or unsubstituted heterocycloalkyl is a substituted orunsubstituted 3 to 8 membered heterocycloalkyl. Further to any compoundand embodiment thereof disclosed herein, in embodiments L¹ and/or L² isindependently a lower substituent, wherein each substituted orunsubstituted alkylene is a substituted or unsubstituted C₁-C₈ alkylene,each substituted or unsubstituted heteroalkylene is a substituted orunsubstituted 2 to 8 membered heteroalkylene, each substituted orunsubstituted cycloalkylene is a substituted or unsubstituted C₃-C₇cycloalkylene, and each substituted or unsubstituted heterocycloalkyleneis a substituted or unsubstituted 3 to 7 membered heterocycloalkylene.

The scintillator-activated photocleavable linker having the structure ofFormula (I) can have the formula:

In Formula Ia, R¹, L¹ and L² are as described herein, includingembodiments thereof. Further to any compositions including the structureof either of Formulae (I) or (Ia), R¹ can be —NO₂. L² can be—(CH₂)_(n3)—O—C(O)—, wherein n3 is an integer from 0 to 5, e.g., 0, 1,2, 3, 4, or 5. n3 can be 1.

Further to any compositions including the structure of either ofFormulae (I) or (Ia), L² can be bound to the chemical agent moiety. Inembodiments, L² forms part of the chemical agent moiety

Further to any composition disclosed herein, the chemical agent moietycan be covalently bound to the scintillator-activated photocleavablelinker through an amine group on the scintillator-activatedphotocleavable linker thereby forming an —NH— connecting moiety. Theterm “—NH— connecting moiety” and the like refer, in the usual andcustomary sense, to a second amine acting as a covalent linkage betweensubstituents on the amine nitrogen. Alternatively, the chemical agentmoiety includes an —NH— group that serves as the point of attachment ofthe chemical agent moiety to the scintillator-activated photocleavablelinker (e.g. the L² group). Thus, upon cleavage this —NH-groups becomesan —NH₂ group attached to the remainder of the released drug (e.g., asin the Doxorubicin example provided herein).

Further to any composition disclosed herein, the chemical agent moietycan be a drug moiety, a hormone moiety, a metal moiety, aradioprotective moiety, a cement moiety, a nucleotide triphosphatemoiety, a protein moiety, a polysaccharide moiety, a neurotransmittermoiety, an enzyme moiety, a tissue factor moiety or a detectable moiety.The chemical agent moiety can be a drug moiety. The chemical agentmoiety can be a hormone moiety, as known in the art. The chemical agentmoiety can be a metal moiety, e.g., a metal or metal chelate incombination with a metal. The chemical agent moiety can be aradioprotective moiety, as known in the art. The chemical agent moietycan be a cement moiety, as known in the art. Cement moieties in thiscontext can be multivalent reactive species which can bind two or morecomponents to be cemented together. The chemical agent moiety can be anucleotide triphosphate moiety, as known in the art. The chemical agentmoiety can be a protein moiety, as known in the art. Exemplary proteinmoieties in this context can include antibodies, protein hormones, andcytokines and other cell signaling proteins, as known in the art. Thechemical agent moiety can be a polysaccharide moiety, as known in theart. The chemical agent moiety can be a neurotransmitter moiety, asknown in the art. The chemical agent moiety can be an enzyme moiety, asknown in the art. The chemical agent moiety can be a tissue factormoiety, as known in the art. The chemical agent moiety can be adetectable moiety. When bound to the nanocrystal scintillator, the drugmoiety can be a prodrug moiety. The drug moiety can be an anticancerdrug moiety or an antibiotic drug moiety. The drug moiety can be ananticancer drug moiety. The term “anticancer drug moiety” and the likerefer, in the usual and customary sense, to a drug moiety found usefulin the treatment or amelioration of cancer, as known in the art. Thedrug moiety can be an antibiotic drug moiety, as known in the art.

An exemplary chemical synthesis scheme for a yttrium oxide scintillatornanocrystal is set forth in Scheme 1 following. In the scheme, anaminosilane modified yttrium oxide nanoparticle (“Y₂O₃”) having ascintillation activator is shown having aminosilane groups emanatingfrom the surface. Reaction of doxorubicin-NHCO conjugated with NHS esterof para-nitrophenylate with the free amine group affords thescintillator nanocrystal linked to a chemical agent moiety through ascintillator-activated photocleavable linker. Subsequent irradiation(“Radiation”) of the scintillator nanocrystal affords a photon which isabsorbed by the scintillator-activated photocleavable linker causingcleavage. The resulting species are Doxo (containing the resultant —NH₂moiety), CO₂, and the scintillator nanocrystal now attached to theremnants of the photocleavage linker.

Pharmaceutical Compositions

In another aspect, there is provided a pharmaceutical compositionincluding a composition including a scintillator nanocrystal linked to achemical agent moiety through a scintillator-activated photocleavablelinker, as disclosed herein, in combination with a pharmaceuticallyacceptable excipient (e.g., carrier).

Suitable pharmaceutically acceptable excipients include, for example,pharmaceutically, physiologically, acceptable organic or inorganiccarrier substances suitable for enteral or parenteral application thatdo not deleteriously react with the scintillator nanocrystal linked to achemical agent moiety through a scintillator-activated photocleavablelinker, or any components thereof.

Suitable pharmaceutically acceptable carriers include water, saltsolutions (such as Ringer's solution), alcohols, oils, gelatins, andcarbohydrates such as lactose, amylose or starch, fatty acid esters,hydroxymethycellulose, and polyvinyl pyrrolidine. Such preparations canbe sterilized and, if desired, mixed with auxiliary agents such aslubricants, preservatives, stabilizers, wetting agents, emulsifiers,salts for influencing osmotic pressure, buffers, coloring, and/oraromatic substances and the like that do not deleteriously react withthe compounds described herein.

Formulations

The compositions described herein can be prepared and administered in awide variety of oral, parenteral, and topical dosage forms. Thus, thecompounds described herein can be administered by injection (e.g.,intravenously, intramuscularly, intracutaneously, subcutaneously,intraduodenally, or intraperitoneally). Also, the compounds describedherein can be administered by inhalation, for example, intranasally.Additionally, the compounds described herein can be administeredtransdermally. It is also envisioned that multiple routes ofadministration (e.g., intramuscular, oral, transdermal) can be used toadminister the compounds described herein. Accordingly, pharmaceuticalcompositions comprising a pharmaceutically acceptable carrier orexcipient and one or more compounds are contemplated.

For preparing pharmaceutical compositions, pharmaceutically acceptablecarriers can be either solid or liquid. Solid form preparations includepowders, tablets, pills, capsules, cachets, suppositories, anddispersible granules. A solid carrier can be one or more substances thatmay also act as diluents, flavoring agents, binders, preservatives,tablet disintegrating agents, or an encapsulating material.

In powders, the carrier is a finely divided solid in a mixture with thefinely divided active component. In tablets, the active component ismixed with the carrier having the necessary binding properties insuitable proportions and compacted in the shape and size desired.

The powders and tablets preferably contain from 5% to 70% of thescintillator noncrystal composition. Suitable carriers are magnesiumcarbonate, magnesium stearate, talc, sugar, lactose, pectin, dextrin,starch, gelatin, tragacanth, methylcellulose, sodiumcarboxymethylcellulose, a low melting wax, cocoa butter, and the like.The term “preparation” is intended to include the formulation of theactive compositions with encapsulating material as a carrier providing acapsule in which the active component with or without other carriers, issurrounded by a carrier, which is thus in association with it.Similarly, cachets and lozenges are included. Tablets, powders,capsules, pills, cachets, and lozenges can be used as solid dosage formssuitable for oral administration.

For preparing suppositories, a low melting wax, such as a mixture offatty acid glycerides or cocoa butter, is first melted and the activecomponent is dispersed homogeneously therein, as by stirring. The moltenhomogeneous mixture is then poured into convenient sized molds, allowedto cool, and thereby to solidify.

Liquid form preparations include solutions, suspensions, and emulsions,for example, water or water/propylene glycol solutions. For parenteralinjection, liquid preparations can be formulated in solution in aqueouspolyethylene glycol solution.

When parenteral application is needed or desired, particularly suitableadmixtures are injectable, sterile solutions, preferably oily or aqueoussolutions, as well as suspensions, emulsions, or implants, includingsuppositories. In particular, carriers for parenteral administrationinclude aqueous solutions of dextrose, cyclodextrins, saline, purewater, ethanol, glycerol, propylene glycol, peanut oil, sesame oil,polyoxyethylene-block polymers, and the like. Ampoules are convenientunit dosages. The compositions described herein can also be incorporatedinto liposomes or administered via transdermal pumps or patches.Pharmaceutical admixtures suitable for use include those described, forexample, in PHARMACEUTICAL SCIENCES (17th Ed., Mack Pub. Co., Easton,Pa.) and WO 96/05309, the teachings of both of which are herebyincorporated by reference.

Aqueous solutions suitable for oral use can be prepared by dissolvingthe active component in water and adding suitable colorants, flavors,stabilizers, and/or thickening agents as desired. Aqueous suspensionssuitable for oral use can be made by dispersing the finely dividedactive component in water with viscous material, such as natural orsynthetic gums, resins, methylcellulose, sodium carboxymethylcellulose,and other well-known suspending agents.

Also included are solid form preparations that are intended to beconverted, shortly before use, to liquid form preparations for oraladministration. Such liquid forms include solutions, suspensions, andemulsions. These preparations may contain, in addition to the activecomponent, colorants, flavors, stabilizers, buffers, artificial andnatural sweeteners, dispersants, thickeners, solubilizing agents, andthe like.

The pharmaceutical preparation can be in unit dosage form. In such formthe preparation is subdivided into unit doses containing appropriatequantities of the active component. The unit dosage form can be apackaged preparation, the package containing discrete quantities ofpreparation, such as packeted tablets, capsules, and powders in vials orampoules. Also, the unit dosage form can be a capsule, tablet, cachet,or lozenge itself, or it can be the appropriate number of any of thesein packaged form. In embodiments, the unit dosage form can be in theform of an applicator pre-filled with a pharmaceutical compositiondescribed herein (for example, a pharmaceutical composition thatcontains an effective amount of a scintillator nanocrystal linked to achemical agent moiety through a scintillator-activated photocleavablelinker). In embodiments, the pre-filled applicator can be filled with apharmaceutical composition in the form of a cream, a gel or an ointmentthat contains a compound described herein.

The quantity of active component in a unit dose preparation may bevaried or adjusted from 0.1 mg to 10000 mg, more typically 1.0 mg to1000 mg, most typically 10 mg to 500 mg, according to the particularapplication and the potency of the active component. The compositioncan, if desired, also contain other compatible therapeutic agents.

Some compounds may have limited solubility in water and therefore mayrequire a surfactant or other appropriate co-solvent in the composition.Such co-solvents include: Polysorbate 20, 60, and 80; Pluronic F-68,F-84, and P-103; cyclodextrin; and polyoxyl 35 castor oil. Suchco-solvents are typically employed at a level between about 0.01% andabout 2% by weight.

Viscosity greater than that of simple aqueous solutions may be desirableto decrease variability in dispensing the formulations, to decreasephysical separation of components of a suspension or emulsion offormulation, and/or otherwise to improve the formulation. Such viscositybuilding agents include, for example, polyvinyl alcohol, polyvinylpyrrolidone, methyl cellulose, hydroxy propyl methylcellulose,hydroxyethyl cellulose, carboxymethyl cellulose, hydroxy propylcellulose, chondroitin sulfate and salts thereof, hyaluronic acid andsalts thereof, and combinations of the foregoing. Such agents aretypically employed at a level between about 0.01% and about 2% byweight.

The compositions may additionally include components to providesustained release and/or comfort. Such components include high molecularweight, anionic mucomimetic polymers, gelling polysaccharides, andfinely-divided drug carrier substrates. These components are discussedin greater detail in U.S. Pat. Nos. 4,911,920; 5,403,841; 5,212,162; and4,861,760. The entire contents of these patents are incorporated hereinby reference in their entirety for all purposes.

Effective Dosages

Pharmaceutical compositions include compositions wherein the activeingredient is contained in a therapeutically effective amount, i.e., inan amount effective to achieve its intended purpose. The actual amounteffective for a particular application will depend, inter alia, on thecondition being treated, as judged by a practioner in the medical orveterinary arts. For example, when administered in methods to treatcancer, such compositions will contain an amount of active ingredienteffective to achieve the desired result (e.g., decreasing the number ofcancer cells in a subject).

The dosage and frequency (single or multiple doses) of compoundadministered can vary depending upon a variety of factors, includingroute of administration; size, age, sex, health, body weight, body massindex, and diet of the recipient; nature and extent of symptoms of thedisease being treated; presence of other diseases or otherhealth-related problems; kind of concurrent treatment; and complicationsfrom any disease or treatment regimen. Other therapeutic regimens oragents can be used in conjunction with the methods and compoundsdescribed herein.

Dosages may be varied depending upon the requirements of the patient andthe compound being employed. The dose administered to a patient shouldbe sufficient to effect a beneficial therapeutic response in the patientover time. The size of the dose also will be determined by theexistence, nature, and extent of any adverse side effects. Generally,treatment is initiated with smaller dosages, which are less than theoptimum dose of the compound. Thereafter, the dosage is increased bysmall increments until the optimum effect under circumstances isreached.

Dosage amounts and intervals can be adjusted individually to providelevels of the administered compound effective for the particularclinical indication being treated. This will provide a therapeuticregimen that is commensurate with the severity of the individual'sdisease state.

Utilizing the teachings provided herein, an effective prophylactic ortherapeutic treatment regimen can be planned that does not causesubstantial toxicity and yet is entirely effective to treat the clinicalsymptoms demonstrated by the particular patient. This planning shouldinvolve the careful choice of active compound by considering factorssuch as compound potency, relative bioavailability, patient body weight,presence and severity of adverse side effects, preferred mode ofadministration, and the toxicity profile of the selected agent.

Toxicity

The ratio between toxicity and therapeutic effect for a particularcompound is its therapeutic index and can be expressed as the ratiobetween LD₅₀ (the amount of compound lethal in 50% of the population)and ED₅₀ (the amount of compound effective in 50% of the population).Compositions that exhibit high therapeutic indices are preferred.Therapeutic index data obtained from cell culture assays and/or animalstudies can be used in formulating a range of dosages for use in humans.The dosage of such compounds preferably lies within a range of plasmaconcentrations that include the ED₅₀ with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized. See, e.g., Fingl etal., In: THE PHARMACOLOGICAL BASIS OF THERAPEUTICS, Ch. 1, p. 1, 1975.The exact formulation, route of administration, and dosage can be chosenby the individual physician in view of the patient's condition and theparticular method in which the compound is used.

Methods of Use

In another aspect, there is provided a method for delivering a chemicalagent moiety to a target site. The method includes: (i) providing acomposition including a scintillator nanocrystal linked to a chemicalagent moiety through scintillator-activated photocleavable linker asdisclosed herein to a location at or near a target site and (ii)cleaving the chemical agent from the remainder of the composition byexposing the composition to radiation thereby delivering the chemicalagent to the target site. The term “target site” and the like refer, inthe usual and customary sense, to a site which can benefit by localadministration of the chemical agent moiety. In medical applications,the chemical agent moiety can be a drug moiety, a hormone moiety or adetectable moiety.

In another aspect, there is provided a method of delivering a chemicalagent moiety to a subject. The method includes: (i) administering acomposition including a scintillator nanocrystal linked to a chemicalagent moiety through scintillator-activated photocleavable linker asdisclosed herein to the subject, and (ii) cleaving the chemical agentfrom the remainder of the compound by exposing the composition toradiation thereby delivering the chemical agent to the subject.

The subject can be a a cancer patient and the chemical agent moiety canbe an anticancer drug agent, and the composition can be administered tothe subject in a therapeutically effective amount.

Other Aspects

The following definitions and embodiments apply to only to the compoundsdisclosed in this section (i.e. section IV) and embodiments P1 to P5listed below.

In a first aspect, there is provided a radiation activated nanoparticleassembly comprising a multilayered nanoparticle in external contact witha lipid-proreagent. The multilayered nanoparticle includes ascintillator core, an inert hydrophilic shell, and an external lipidbilayer. The lipid-proreagent includes a head group, a plurality of tailgroups, and a proreagent attached at the head group with aphotocleavable linker.

In embodiments, of the radiation activated nanoparticle assembly, theplurality of tail groups contact the external lipid bilayer.

In embodiments, the proreagent is released from the lipid-proreagent byscission of the photocleavable linker.

In embodiments, scission results from release of light from thescintillator core in response to radiation impinging thereon.

The terms “radiation activated nanoparticle assembly” and the like referto compounds and assemblies described for Section IV wherein amultilayered nanoparticle is associated with a lipid-proreagent. Theassociation of multilayered nanoparticle with the lipid-proreagent canbe covalent, e.g., covalent bonding to the external lipid bilayer of themultilayered nanoparticle, or noncovalent, e.g., association with theexternal lipid bilayer of the multilayered nanoparticle byphysiochemical processes known in the art, e.g., electrostatic, van derWaals, hydrophobic interaction, and the like.

The terms “prodrug,” “prodrug moiety,” “proreagent” and the like areused according to the plain ordinary meaning and is intended torepresent covalently bonded carriers, which are capable of releasing anactive ingredient (e.g., a drug) when the prodrug is administered to asubject. Accordingly, the terms “proreagent” and the like in thiscontext refer to compounds which are covalently bound to a carrier(e.g., lipid moiety) via a linker, e.g., a photocleavable linker.Cleavage of the photocleavable linker results in release of the reagent.In one embodiment, the proreagent includes a therapeutic agent, abiodistribution agent, or a labeling agent. Thus, the term “proreagent”in the context of the release of a therapeutic drug, is synonymous withthe term “prodrug.” The term “lipid-proreagent” in the context ofcompounds disclosed herein refers to a proreagent bound to a lipidthrough a photocleavable linker. The terms “biodistribution agent” andthe like refer, in the customary sense, to chemical species which canmodulate the distribution of the radiation activated nanoparticleassembly within a subject, as known in the art, e.g., polyethyleneglycol, as described herein. The terms “labeling agent” and the likerefer, in the customary sense, to chemical species which can provide adetectable signal, e.g., fluorescent or colored dye, radioactiveemitter, spin labeling reagent, and the like.

Example 1 (Section IV). Overall Research Design and Scope Example 1.1(Section IV). Preparation of Scintillator Nanocrystals

Preparation of scintillator nanocrystals of various compositions andquantification of their quantum light yield, between 360 and 380 nm, canbe demonstrated in response to a range of radiation doses and energies.Our target is to attain 28,000-46,000 photons/MeV at doses ≤2 Gy whichshould be attainable as scintillators used for dosimetry robustly emitat low doses.

Example 1.2 (Section IV). Quantum Yield Quantification of ScintillatorsAccording to Radiation Dose

Nanopowdered scintillators can be exposed to X-rays between 0.1 and 10Gy (1 Gy increments, 2 Gy/minute) and energies between 250 KeV and 6MeV. The scintillator with the greatest photon yield at low doses (0.1-2Gy) can be selected for further development as a nanoparticle. Photonemission of ≥28,000 photons at 2 Gy or less is the goal.

Example 1.3 (Section IV). Encapsulation of Scintillators

Encapsulation of the most efficient scintillator with a hydrophilicshell which in turn can be coated with a lipid bilayer is desired. SeeFIG. 1. Lipids in this bilayer can be chemically linked to a cancerdrug, e.g., doxorubicin (Dox). Dox potently suppresses humanglioblastoma (GBM) cells in culture, but clinically it causes acute andlong term toxicity, which is dose-limiting, so that only a portion ofthe IC90 dose used in vitro can in the clinical setting be delivered tobrain tumors. Linking Dox to a nanocrystal can render it inactive andnon-toxic so that high doses may be administered, which in turn canfacilitate the accumulation of significant concentrations of the drugwithin the tumor. The accumulated Dox can then be activated by localizedirradiation only in the vicinity of brain tumors.

Example 1.4 (Section IV). Connecting Linker

Exemplary linkers connecting a cancer drug, e.g., Dox, to the lipidcoated nanocrystal have been designed, synthesized, and tested to bedisrupted by ultraviolet (UV) light at 360 nm. This linker can beadapted for the coated nanocrystal platform (FIG. 1). This linkerbreakage wavelength was selected because at wavelengths much longer than350-360 nm the light will not have sufficient energy to break bonds, andat shorter wavelengths photo-induced damage of the drug will begin tooccur.

Example 1.5 (Section IV). Rate and Extent of Drug Release

The rate and extent of Dox release with irradiation can be measured andquantified. The potency of the nanocrystal-Dox platform, with/outactivating radiation, against human GBM grown in culture can be comparedto Dox in free form, and in the context of integrin-based tumortargeting versus non-targeting.

Example 1.6 (Section IV). Maximum Tolerated Dose (MTD)

The maximum tolerated dose (MTD) of free Dox and nanocrystal bound Doxcan be assessed with IV tail vein injection, employing protocols wellknown in the art.

Example 1.7 (Section IV). In Vitro Potency Again Primary HumanGlioblastoma Cells in Culture

In vitro potency of scintillator nanocrystal-Dox platform againstprimary human glioblastoma cells in culture can be determined.Established and primary human GBM cell lines grown in vitro can eitherhave the scintillator nanocrystal-Dox platform added, free Dox,nanocrystals only, and can receive a range of radiation doses or noradiation. Survival of the cells can be measured and comparedstatistically.

Example 1.8 (Section IV). Brain Entry, Biodistribution and Toxicity

Without wishing to be bound by any theory, it is believed thatnanoparticles can enter the brain tumors via leaky tumor microvessels.In addition recent reports indicate that PEGylated liposomes bearing Doxcross the blood brain barrier (BBB) and that transferrin ligand (iron)significantly potentiated the movement of liposomes across the BBB. Forthese reasons related to the ability to permeate tumors and penetratethe BBB, the nanoparticles can incorporate these moieties, and theinfluence of size on BBB permeation can be assessed using variousdiameters of nanoparticles, e.g., 100-150 nm and 50 nm.

Biodistribution is an important issue for eventually moving ahead toclinical studies with nanoparticles, although the non-toxic nature ofthe nanocrystal-Dox prodrug can reduce side effects to major organsassuming the Dox is stably attached to the nanocrystal in the absence ofradiation. The in vivo distribution of fluorescent nanocrystals and theyield of Dox after a single injection of free drug or nanocrystal boundDox followed by irradiation, can be determined for a primary human GBMgrowing orthotopically, and for normal brain, heart, lung, spleen, liverand kidneys. The fluorescent nanocrystals can allow identification oftheir location and density, and we can determine the degree ofcorrelation with precise LC mass spectroscopy measurements of actual Doxconcentrations in all tissue samples. All organ samples can be examinedhistologically for any signs of pathology to provide preliminarytoxicity data.

Example 1.9 (Section IV). In Vivo Anti-GBM Efficacy

One week after the mice are stereotaxically implanted with tumors theycan be injected with free Dox or nanocrystal (50 or 100-150 nm) boundDox at the previously determined MTD, or nanocrystal alone, then headonly or sham irradiated with 1, 2, or 4 GY at four hours afterinjection. We can use a head only irradiation mouse holder apparatus.There can be two treatments, once a week for two weeks, and then fourdays after the last treatment. The mice can be sacrificed and the brainand major organs removed. The brain can be sectioned and the tumorvolume measured. The mice can be weighed weekly prior to sacrifice.Major organs can be examined by histopathology for evidence of damage.

Example 2 (Section IV). Fabrication and Quantification of RadiationScintillators Example 2.1 (Section IV). Fabrication Methodology andOptimizing Light Output

Scintillator nanocrystals 100-150 nm diameter can be prepared with aphoton yield of 28,000-46,000 photons/MeV and emission peaks between330-365 nm under T-ray excitation using cerium activated halidecompositions such as K₂LaCl₅, BaF₂, RbGd₂Br₇ and LaC₁₃. The mostimportant scintillation mechanism is energy transfer by directelectron-hole recombination on the cerium site23. These scintillatorscan be prepared by various synthetic methods15, but for fabrication ofcore-shell nano-sized particles, optimized low-temperature hydrothermaland co-precipitation methods are believe to be well suited.

The challenge with nano-sized luminescent particles is the reducedquantum efficiency due to poor absorption of the excitation radiationarising from pronounced reflectance losses coupled with nonradiativerelaxation at the surface states (as demonstrated in FIG. 3—the signalto noise ratio is small). To alleviate these problems, core/shellnanostructures can be used to stabilize the surface of thenanoparticles. FIG. 3 shows a transmission electron micrograph andradioluminescence spectrum of single core BaF₂:Ce³⁺ prepared byco-precipitation. Note the emission peak at the required wavelength of360 nm. This co-precipitation method was adapted to create core-shellstructures as shown in FIG. 3. The photoluminescence emission intensitywas 50% higher for core/shell Y₂O₃:Eu3+/SiO2 compared to bare coreparticles (FIG. 4). This strategy can be extended to emitters in the330-360 nm UV range.

Briefly, the core/shell nanocrystals can be prepared by a Pechinisol-gel process, by dissolving in a nitric acid solution (volume ratioof water and nitric acid=1:1) to form an aqueous solution. Subsequently,4.2 g citric acid which acts as chelating agent for metal ions, and 2.23ml ethylene glycol (EG) can be introduced (molar ratio ofmetal:CA:EG=1:1:2). Then 5 g polyethylene glycol (PEG) as across-linking agent can be added into the aqueous solution to effect aPEG concentration of 0.05 g/ml. The solution can be continuously stirredfor 10 h at 80° C. to form a transparent gel. This gel can be preheatedat 300° C. for 1 h, and then calcined at 800° C. for 1 h in a furnace inair to remove organic materials and to obtain the nanophosphor powders.

Example 2.2 (Section IV). Coating Scintillator Nanocrystals with anInert Hydrophilic Surface

Inert silica can be applied to allow later coating with the lipidbilayer. This process can be started with the hydrolysis of TEOS inalcohol, water, and ammonia. First, 1 g of the core particles can beadded to 30 ml 1-propanol. The mixture can be agitated usingultrasonification for 1.5 h to disperse the core particles. Then, 0.5 mlof deionized water, 0.4 ml NH4OH and 0.1 ml TEOS can be added. The ratiobetween the concentrations of core particles and reagents (H₂O, NH₄OHand TEOS) can be adjusted to avoid self-nucleation of silica and thus,the formation of core-free silica spheres. The reaction can be continuedin the ultrasonicator to obtain more dispersed core and uniform SiO₂coating on the core. In order to prevent heating of the solution and tobetter disperse the nanoparticles, ice can frequently be added intoultrasonification bath and the bath temperature fixed at 20° C.Centrifugation can separate reaction products from the suspension andrinsed with ethanol four times. The concentration of H₂O and NH4OH inthe 1-propanol solution may be controlled, instead of lengthening thedeposition time, because the change of concentration of H2O and NH4OHhas been noted as a comparatively efficient way to deposit thicker SiO2shells.

Example 2.3 (Section IV). Quantum Efficiency of Scintillators

The quantum light yield with radiation exposure (1-10 Gy) can bemeasured with known quantities of nanocrystals in a cuvette andmeasuring light output with an ultrasensitive CCD array. This work canbe performed by adjusting X-ray dose and energy directed at a samplecuvette for materials surface studies. A sensitive CCD detector can bepositioned to capture nanopowdered scintillator emission in response to0.5, 1, 2, 3, 4, 5, 6, 7, and 8 Gy of X-rays applied at energies of 250KeV to 2, 4 and 6 Mev. The acquired data can be adjusted to account for47 (all directions) light emission and, based on the quantity ofscintillator material, the quantum yield can be calculated. Thescintillator can be selected that generates the most photon yield at lowdoses, and preferably lower energies.

Example 3 (Section IV). Nanocrystal-Dox Prodrug Platform AssemblyExample 3.1 (Section IV). Nanocrystal Lipid Coating

The most efficient scintillator nanocrystal can be surface modified witha variously hydrophilic, inert material to facilitate the attachment offunctionalized lipid components in a bilayer. Thesurface-functionalization method of the scintillator nanocrystal canutilize alkoxysilane molecules with hydrophilic amino groups at theend—3-aminopropyltriethoxysilane, NH₂(CH₂)₃Si(EtO)₃, (3-APTES) (>98%,Alfa Aesar). Eu powders can be dispersed in acetone under mildagitation. Subsequently, 5 vol % 3-aminopropyltriethoxysilane can beadded drop-wise, and the solution can be magnetically stirred at theboiling temperature of acetone (56° C.) and under flowing N2 gas for 180minutes. The solution can be centrifuged and the particles that remaincan be washed with acetone and dried in air. These particles can then bedispersed in deionized (DI) water. The lipid bilayer is described inTable 1 and can include multiple types of lipids, e.g., 1, 2, 3, or moretypes, as well as (i) polyethylglycol moieties, (ii) UV radiationcrosslinkable acetylene groups, and (iii) synthetic αvβ3/α5β1 receptorligands (Table 1). The relative surface composition of the resultantnanoparticles can be similar to what we have successfully used inprevious studies for other applications.

TABLE 1 Proposed nanocrystal external lipid layer constituents.Constituent Percent of Outer Shell Fluor (Rhodamine) 2 DSPC- PEG 2000 10UV Crosslinkable acetylated DOPE 5 αvβ3/α5β1 ligand (-PEG-DOPE) 5Cholesterol 10 DOPE 30 DSPC 28 DOPE = dioleophosphadylethnalolamine DSPC= distearoylphophatylcholineBODIPY=6-(4,4-difluoro-5-(2-thienyl-4-bora-3a,4a-diaza-s-indacene-3-yl)styryloxy-acetyl)aminohexamido-DOPE

Example 3.2 (Section IV). Integrin Targeting Ligand and FluorescentMarker Attachment

Covalently attached to the lipid bilayer can be an antitumor drug, e.g.,Dox, αvβ3 integrin tumor/tumor vessel targeting ligands (cyclic RGD[Arginylglycylaspartic acid]-containing peptides), fluorescent reporter(BODIPY), and/or stealth components (polyethylene glycol [PEG]) for longcirculation times. Dox can be linked to the lipid layer using aphotosensitive linker (i.e., photocleavalbe linker). In order to attachthe RGD tumor (integrin) targeting cyclic peptide, the peptide caninitially be conjugated to a short linker, succinimidylester-(PEO)4-maleimide, and simultaneously DSPE can be reacted withiminothiolane to produce a free thiol. The DSPE containing the freethiol group can be reacted with the peptide-(PEO)4-maleimide to producethe peptide-lipid conjugates, and these conjugates can be recrystallizedin methanol/diethyl ether 1:9 at 4° C. overnight. The exact mass of thepeptide-lipid conjugates can be verified by mass spectroscopy.

For BODIPY fluorescent marker conjugation to the external DOPE lipidlayer, BODIPY succinamidyl ester can be dissolved in DMSO, to which canbe added triethylamine and DOPE dissolved in chloroform. The reactionmixture can be stirred for 60 min, and the solvent removed. The dryresidue can be dissolved in chloroform.

Example 3.3 (Section IV). Diameters of Nanoparticles

Various sizes of nanoparticles can be made and evaluated, e.g., a100-150 nm diameter group for primary tumors and metastatic sites, and asecond group of 50 nm diameters with transferrin ligand attached forgeneral brain penetration across the blood brain barrier (BBB).Nanoparticle structural optimization can be guided by charge/sizemeasurements (Malvern Zetasizer) and electron microscopy (see below).

Without wishing to be bound by any theory, it is believed thatultimately from a clinical standpoint the larger particles willprimarily be intended to deliver a significant drug payload to primarytumors and vascularized metastatic foci, while the smaller 50 nmparticles are intended to more readily penetrate the BBB, release theirpayload after carefully directed irradiation, and the free drug willengage poorly vascularized, or less leaky tumor areas, as well asinfiltrating single cells in areas or normal brain. The behavior andefficacy of nanoparticles having different particle size can be assessedin an animal model.

Example 3.4 (Section IV). Photosensitive Linkage of Dox to theNanocrystal Lipid Coating

This part of the nanoparticle assembly sequence lies at the heart ofthis potentially transformative technology. The ability to efficientlyrelease Dox previously bound to be non-toxic can be triggered by UVlight, e.g., at approximately 350-360 nm. The chemical linker can be thesame one we have used to make a light sensitive Dox prodrug, except thatfor the presently proposed research the linker can attach Dox to thelipid surface of the scintillator nanocrystal.

To prepare the linkage Dox can be dissolved in DCM and with lipid coatednanocrystals, and can be added to 1-(3-nitrophenyl) ethylcarbonochloridate in DCM at 1:1 molar ratio. The reaction mixture can bestirred vigorously with a magnetic Teflon-coated stir bar for 30 min.The linked drug product can be purified by HPLC semiprep purification.Without wishing to be bound by any theory, it is believed that theoverall yield of linked drug can be up to 70% or more, 70%, 80%, 90% ormore, and that the linkage will be stable at neutral pH.

Releasing free drug from the nanocrystal conjugate is based upon thephotocleavable characteristic of the nitrophenyl group. The mechanism ofphotolysis has been adapted from established mechanisms and is shownschematically in FIG. 2. Light absorption at 365 nm causes the electronconfigurations in the nitrophenyl group to rearrange, inducing theformation of an unstable 6-membered ring with one of the nitro group'soxygen atoms. The destabilization and rearrangement of this ring causesthe cleavage, releasing the carbamate group along with drug. Thecarbamate undergoes hydrolysis in aqueous conditions, producing CO2 andfree drug. This linker can attach Dox to the nanocrystal lipid coating.

Example 3.5 (Section IV). Integrin Targeting Ligand Structure andSynthesis

The tumor and tumor vasculature targeting component of the liposome canbe synthesized by the reaction of a peg activated-DOPE lipid withreactive covalently linkable αvβ3/a5b1 ligand, shown in FIG. 5.

Example 4 (Section IV). Physical Characterization of ScintillatorNanoparticle-Dox Platform Example 4.1 (Section IV). Size, Charge andMorphology

The nanoparticle suspension can be diluted in 1/10 in MilliQ water, and100 μl of the dilution sized with a Zetasizer using lightbackscattering. The same instrument can be used to measure the particlenet charge expressed in mV. Morphology and size can be furthercharacterized using scanning electron microscopy (SEM). Samples can beprepared by dropping 5 μl of particle suspension onto a polished siliconwafer. After drying the droplet at room temperature overnight, thesample can be coated with chromium and then imaged by scanning electronmicroscopy (SEM).

Example 4.2 (Section IV). Evaluation of Doxorubicin (Dox) AttachmentEfficiency

For the assessment of the Dox content the nanocrystal-Dox platform canbe dissolved in DMSO containing 0.004% HCl. After ultrasonication for 20min, the insoluble material can be separated by centrifugation for 15min at 16,000 g. The concentration of Dox in the supernatant can bemeasured spectrophotometrically at 480 nm. The attachment efficiency(percentage of Dox bound to nanocrystals) can be calculated as thedifference between the initial drug content and the amount of free Doxin the filtrate after separation of the nanoparticles byultrafiltration.

Example 4.3 (Section IV). Doxorubicin Release from ScintillatorNanocrystals without Irradiation

The stability of the nanocrystal-Dox platform and the kinetics of Doxrelease from the nanocrystals can be investigated in aqueous milieu.Non-irradiated nanocrystal-Dox can be added to Milli-Q water and therelease at 1, 4, 6 8, and 12 hours quantified. For the irradiation teststhe nanoplatform can be suspended in Milli-Q water, and diluted 25-foldwith water. Then the diluted suspension can be incubated at 37° C. underconstant stirring at 150 rpm and irradiated with X-rays at dosesincluding 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 Gy at 2 Gy/min and atbetween 250 KeV and 6 MeV. For analysis 3 ml aliquots of the irradiatedand non-irradiated suspensions can be centrifuged (20,000 g for 30minutes at ambient temperature) to separate the nanocrystals. Theconcentration of Dox in the supernatant can be measured byspectrophotometry at lambda max=480 nm.

Example 5 (Section IV). In Vitro Potency of Radiation ActivatableNanocrystal-Dox Platform

The nanocrystal-drug platform and free Dox can be suspended inphysiological saline and added to both established and primary humanglioblastoma cell lines (U87, GBM4/8) grown in cell culture, with RPMImedium supplemented with antibiotics, at 370 C and with 30% CO2. Two andfour hours after addition of the nanoparticles or drug, separate groupsof flasks can be irradiated at doses ranging from 0.5, 1, 2, 3, 4, 5, 6,7, 8, 9, Gy of X-rays at 2 Gy/min. The test conditions can be:Irradiation alone, irradiation and free drug, irradiation andnanocrystal only, free drug only, irradiation and nanocrystal with bounddrug, integrin targeted nanocrystal (50 vs. 150 nm) with bound drug, andnon-targeted nanocrystal (50 nm vs. 150 nm) with bound drug.

Twenty-four hours after irradiation the cells can be washed withphosphate buffered saline, replenished with fresh medium, and culturedfor an additional 6 days. Cell colonies can be fixed with ethanol,stained with Giemsa, and the cell population expressed as a percentageof the non-irradiated, non-drug controls.

Statistical Analysis: In vitro GBM cell killing by theradiation-activated nanoparticle-drug can be assessed by dividing theaverage number of viable cells from three plate replicates (pernanoparticle and radiation dose level), by the average of threecontrols. At a type I error rate of 0.05, using a one-sided t-test, wewill have 80% power to evaluate whether a decrease in mean percentviable cells is significantly lower than 100%, conservatively assumingstandard deviation percent viable cells is 15%.

Example 6 (Section IV). In Vivo Tumor Accumulation and Kinetics of theNanocrystal-Dox Platform Example 6.1 (Section IV). Maximum ToleratedDose and Clearance Kinetics in Health Mice

To determine the in vivo maximum tolerated dose for free and nanocrystalbound DOX (50 nm, 150 nm), both can be administered I.V. to 10 BALB/cmice (total=30 mice) at doses of 0 (PBS), 20, 40, and 60 mg/kg DOXequivalents. The weights and general health of the mice can be monitoreduntil the 14th day after injection, or until mice in the group receivingthe highest dose become lethargic and show obvious signs of morbiditysuch as reduced weight, reduced food intake. At the conclusion of theexperiment all mice can be sacrificed and blood can be collected, serumseparated and analyzed for serum creatine kinase, lactic dehydrogenase,and serum transaminase to determine the presence of damage to muscletissue and to the liver at various dose levels.

Preliminary pharmacokinetic (PK) studies can be performed. Briefly, testmice can be given escalating doses of the fluorescence nanocrystal boundDox. PK studies can be performed on healthy balb/c mice in sets of 8mice per PK variable. Mice can receive the nanocrystal-Dox byintravenous injection, and the PK variables and their acceptable limitscan include; (1) Circulation half-life (t_(1/2)>1 hour), (2) Area underthe curve (AUC >200 hr/ng/ml), (3) Distribution volume (Vss >2× bodywater), rate of elimination (CL >50% of blood flow), (4) the ratio ofAUC (brain)/blood). Groups of 10 mice can receive either 1 mg/kg, 10mg/kg, 20 mg/kg, 30 mg/kg, 40 mg/kg or 50 mg/kg inhibitor intravenouslyvia the tail vein. The brains and plasma of different treated mice canbe removed at 0.1, 0.5, 1, 2, and 4 hours after receiving inhibitor (twomice per dose at each time point), homogenized and a small aliquot canbe removed to measure fluorescence. The homogenate can be spiked with aninternal standard, and subjected to organic extraction and LC-Massspectrometry. A standard curve of concentration/peak area for Dox canserve as a reference and can be correlated with sample fluorescencemeasurements.

Example 6.2 (Section IV). Pulse Dosing

High-dose pulse regimens, transferrin ligand (iron), and PLGA, can beexplored to enhance brain entry of the nanocrystal-Dox platform. Theattendant risk of increased toxicity can be avoided by high dosesadministered for a shorter time period or less frequently. Doses of theMTD, 100 and 300 mg/kg free DOX equivalent can be assessed in terms ofbrain concentration of drug as previously described in a total of 30mice. The brains and plasma of different treated mice can be removed andsubjected to organic extraction and LC-Mass spectrometry to quantify Doxlevels. One goal is to attain a micromolar brain concentration which candemonstrate the need and feasibility of dosing for later preclinical andpossibly human studies.

Example 6.3 (Section IV). Tumor Accumulation and Biodistribution

For these studies primary GBM tissue can be acquired from fresh surgicalspecimens (IRB approval/infrastructure in place) which undergomechanical and enzymatic dissociation for isolation and in vitro cultureof tumor-initiating stem cells. The dissociated tissue can be washed,filtered through a 30 m mesh and plated onto ultra-low adherence flasksat a concentration of 500,000 to 1, 500,000 viable cells/ml. The stemcell isolation medium can include human recombinant EGF, human bFGF andheparin. The tumor initiating stem cells routinely grow in suspensionand form spheres comprised of a mix of GBM cell types. Sphere culturescan be passaged by dissociation, washing and resuspension in neural stemcell culture medium. The cells can be concentrated into a compactsuspension (200,000 cells/1 L), and injected stereotaxically into theright frontal cortex of nu/nu mice (3 mm depth). The mice can all berandomized after all of them have been implanted. This modelrecapitulates the natural history of GBM in human patients, exhibitinginfiltrative invasion and an irregular tumor morphology.

The tumor accumulation rate of the integrin-targeted and non-targetedliposomes can be characterized according to nanocrystal fluorescence andLC-Mass spec measurements of Dox. Briefly, at one week after tumorinoculation, the mice can be injected with the lipid coated nanocrystalsin increments of two hours after injection, up to 8 hours, andthereafter at 24 hours, mice can be injected with the vascular markerFITC-lectin, and sacrificed. There can be 30 mice per time point for atotal of 120 (10 targeted and 10 non-targeted receiving nanocrystal Doxat the MTD/pulsed dosing optimum, and 10 mice receiving free Dox at thefree Dox MTD) and at each time point the brain, heart, kidneys, lungs,and spleen can be removed from the cohort of mice. The fluorescenceassociated with the brain tumor can be imaged using a small animalfluorescence imager.

The tumors can be imaged in brightfield transmitted light mode andfluorescence mode at 4×, 10×, 20× and 40× to determine the extent ofnanocrystal associated fluorescence imaged. The images can consist ofsingle focal plane XY frames. The total fluorescence at the tumor sitecan be quantified and normalized as the proportion of illuminated pixelsout of the total image pixels. The other organs can be sectioned andalso imaged and then processed for histology. The resultant fluorescencedata sets for both targeted and nontargeted liposomes can be plotted foreach post-injection time point to show tumor accumulation, and to revealthe optimal time of nanoparticle accumulation at the tumor site to guideradiation exposure. Samples of the tumor, normal brain and other organscan be homogenized, extracted in ethyl acetate, dried and resuspended inwater for LC mass spec. The concentration of Dox per g of tissue can bedetermined and compared statistically with the percentage fluorescenceat each time point. The major organ sections can be examined by ahistopathologist for the signs of tissue damage.

Example 7 (Section IV). In Vivo Efficacy and Toxicity of ScintillatorNanocrystal-Dox Platform

In order to clearly demonstrate any enhanced efficacy and reducedsystemic toxicity provided by the scintillator nanocrystal—Dox prodrugplatform, in vivo experiments with a realistic GBM model and theappropriate controls can be done. Testing can be done using anorthotopic cancer stem cell based GBM model that authenticallyrecapitulates the human disease. As described previously human primaryGBM can be prepared and implanted stereotaxically into the right frontalcortex of nu/nu mice. We can implant 10 mice per group for a total of200 mice and the major treatment injection groups can be: (i) salinecontrols, (ii) coated nanocrystals without liposomes, (iii) free Dox,(iv) coated non-targeted nanocrystals with Doxorubicin, and (vi) coatedtumor/tumor vessel targeted nanocrystals with Dox (50 nm nanoparticlesand with/out transferrin and 100 nm). The previously determined MTD candetermine dosing. The first treatment can be 7 days after tumorimplantation. The mice can be treated intravenously once per week fortwo weeks with the MTD (free Dox and liposomal Dox). At 4 hours afterboth injections, the mice can be lightly sedated with ketamineadministered subcutaneously, and placed in a custom holder with bodyshielding to receive head only gamma or sham irradiation at 2 Gy/minwith 1 or 2 Gy at 1.12 MeV. Three days after the second treatment themice can be deeply anesthetized with Nembutal and a 2 ml blood samplecan be acquired, after which the subjects can be euthanized with CO₂ andthe brain, heart, lungs, spleen, kidney and liver can be removed forhistology. Table 2 shows the set of experiments to be conducted.

TABLE 2 Array of testable conditions using GBM tumor bearing mice. HEADPARTICLE Dose Dox NANOCRYSTAL TARGETED IRRADIATION SIZE (nm) (Gy)Transferrin — — — sham 100 1, 2 — X — — X 100 1, 2 — — X — sham 100 1, 2— — X — X 100 1, 2 — X X — sham 100 1, 2 — X X — X 100 1, 2 — X X X X100 1, 2 — X X X X 100 1, 2 X X X X X 50 1, 2 — X X X X 50 1, 2 X

Example 7.1 (Section IV). Quantitative Measurements of Tumor Responseand Toxicity

The measurements and comparisons listed below can be used to compare theexperimental groups indicated in Table 2:

1—Primary Tumor Response—The tumor volume can be determined from serialH&E slides using commercial software to define the tumor and calculatevolume derived from all slices. The data can be compared statisticallybetween groups.

2—Number of Detectable Metastases—The metastases to surrounding theprimary tumor can be mapped from serial H&E sections. The number andmean size of these foci can be compared between the treated groups.

3—Analysis of proliferation—To assess the proliferating cells,immunohistochemical staining with antibodies against Ki67 can beperformed. The Ki67-labeling index can be expressed as the ratio ofpositively stained tumor cells of all cells determined from at least sixrandom high power fields (40× in each subject).

4—Determination of necrotic areas—For evaluation of necrosis theH&E-stained slides can be examined using a semi-quantitative scoringsystem: 0, no necrotic area; 1, solitary necroses; 2, less than 50% ofthe tumor area occupied by necroses; 3, more than 50% of the tumor cellsper area in the necrotic state.

5—Assessment of Systemic toxicity—All mice can be weighed before tumorimplants, daily during drug treatment, and at experiment conclusion, toreveal any accelerated weight loss due to systemic drug toxicity. Thesamples of blood, lung, liver heart, GI tract, pancreas and kidneys canbe prepared and sent the UCS Cancer Center Histology Core for hematologyand histopathology. The presence, number and severity of anyabnormalities can each be tabulated for every group, and comparedstatistically between groups.

Statistical Analysis. ANOVA models can be built to compare a continuousoutcome (e.g., tumor volume, number of metastases, toxicitymeasures—compared separately) between groups and logistic models tocompare a binary outcome (e.g., a binarized toxicity outcome). Todemonstrate the enhanced efficacy of using targeted nanocrystal-Dox andirradiation within free Dox or nanocrystal-Dox groups among tumorbearing mice, Jonckheere-Terpstra rank-based trend tests can be used forcontinuous outcomes and Cochran-Armitage trend tests for binary outcomesto test for a trend with respect to the subgroups in the same order asthey are listed in Table 2.

Sample size and power. With 10 mice per group, considering thecomparison using targeting and the primary tumor response between Doxand nanocrystal-Dox, i.e., tumor volume, using a two-sided t-test, at atype I error rate of 0.05, we will have 80% power to detect a meandifference of 1.4 standard deviations of the tumor volumes, etc. Othercomparisons, involving main effects or tests of trend across multiplegroups, will have increased power as the sample sizes will be larger.

Example 8 (Section IV). Development of UV-Emitting Nanoscintillators toRelease Prodrugs for Treatment of Brain Tumors: Novel Strategy for DrugDelivery on Specific Sites by Using YAG-Pr Nanoscintillators

Abstract. Ultraviolet emitting YAG-Pr nanoscintillators were produced bycombustion synthesis and post-annealed in air at 1200° C. The X-raydiffraction analysis revealed the formation of the cubic garnet Y₃Al₅O₁₂crystalline structure and TEM revealed the morphology and size ofnanocrystalline clusters that were successfully deagglomerated withultrasonic processing. Praseodymium was used as the impurity dopant inthis complex structure at concentration levels in range of x=0.5˜2.0 at.% that yield strong UV emission band (λ=300˜400 nm) originated from4f5d→³H₄ transitions within the Pr³⁺ electronic levels efficientlyactivated with UV photons of shorter wavelength (λ=292 nm).Radioluminescence experiments were also performed by using high energyx-Ray photons (50 MeV) resulting in a dramatic enhancement of the4f5d→³H₄ transitions. These nanocrystalline praseodymium-doped yttriumaluminates provide promising optical properties to be used asnanoscintillators in modern medicine for the development of novel drugdelivery systems. Thus, it is expected that the UV light activated bythe irradiation can trigger the release/activation of a prodrug.

Introduction. In the last few years, significant progress has beenachieved in the field of nanomedicine and bionanotechnology however,design and development of nanomaterials to be used for reliabledetection of diseases at an early stage and effective drug-delivery attargeted sites are still in development. Obtaining these nanomaterialsin an efficient way represents a tremendous scientific challenge andeffort of research.

Inorganic luminescent nanomaterials are known as nanophosphors. Thesefascinating nanocrystalline compounds posses the property to absorbenergy (UV photons, X-ray or Infrared) and convert it into visiblelight. Phosphors, in general, are composed of a highly stable hostlattice that can be doped with a small concentration of impurity atoms(called activators) that produce the luminescence [Oli 1997]. Phosphorsare inorganic materials with luminescent properties of interest foroptoelectronic devices and radiation detection, including displays,lighting and imaging.

The importance of these class of materials stems mainly form thepossibility of achieving unique properties related to the reduceddimensionality and spatial confinement of the energy states, as seen innanoscale semiconductors [Ref. 1 paper Yukihara], and the possibility ofsynthesis at much lower temperatures when compared to traditionalmethods like solid state reaction and crystal growth.

Scintillation is defined by the flash of light produced in a transparentmaterial by the passage of a particle (electron, alpha particle, an ion,or a high-energy photon). The process of scintillation is one ofluminescence whereby light of a characteristic spectrum is emittedfollowing the absorption of radiation. The emitted radiation is usuallyless energetic than the absorbed.

In the last decade, great research efforts are focused on thedevelopment of new and more efficient scintillation crystals fordetection of ionizing radiation related mainly to imaging systems formedical diagnosis. There is no reports of the use of nanoscintillatorsfor the use in biological systems for drug delivery at specific sites inthe body.

Indeed, there is a great interest in developing nanophosphors forspecific performance in new fields of medicine and biotechnology. Forexample it is desirable to synthesize nanophosphors that emit near-UVlight (λ=300˜400 nm) when exposed to radiation (X-rays or Gamma) forapplications in nanomedicine as drug delivery systems. It is expectedthat the UV light activated by the irradiation can trigger therelease/activation of a prodrug.

The main goal of research in the area of development of new strategiesagainst brain cancer. The design of our proposal is focused on thesynthesis of inorganic compounds that are able to interact with knownprodrugs, with the objective of developing anticancer agents.

Since all anticancer agents interact with both normal cells andcancerous cells, the idea is to develop an anticancer prototype ofaction with much greater specificity. That is, strategies that can bemanipulated just to destroy malignant tumors, without damaging as far aspossible, healthy tissues.

Our model encompasses the development of photosensible nanostructureswith anti-cancer potential. The approach of this design is based in twodifferent mechanisms:

1. UV radiation emission due to luminescent nanoparticles activatedthrough minimum interaction with X-rays or Gamma radiation.

2. Radical decomposition of a formulated prodrug through photolysisprovided by UV photons from the nanophosphor, and induction of cellulardamage by the interaction of the organometallic radicals (prodrug) withthe cancer cells.

Thus, the model implies firstly, the design of organometallic basedprodrug that under physiological condition are harmless for the cells,but that by exposing these clusters to highly energetic radiation, showcytotoxic activity. Secondly, the combination of luminescentnanoparticles with organometallic prodrug can imply the amplification ofcytotoxic effects through the emission of UV photons by activation ofthe nanophosphors under radiation with higher energy in order to inducedamage the malign cells.

Potential advantages of this strategy include: the specificity of themechanism to kill malignant cells and the use of luminescentnanoparticles and organometallic clusters (in absence of radiation)harmless for the healthy tissues, of easy elimination from the biologicsystem due their size (less than 50-60 nm) and permeable traffic throughthe membranes; also the employment of radiation doses, possible lowerthan required in the conventional radiotherapy.

Nanophosphors with the ability to convert X-rays or Gamma rays into UVor visible photons (scintillators) are of interest. There are severalcompositions with optical properties that are suitable as scintillators;however, very few offer the possibility to be used in our approach. Wehave selected Pr-doped yttrium aluminum garnet (Y₃Al₅O₁₂:Pr³⁺) sincethis formula is one of the most chemically stable under energeticradiation (e.g., X-rays or Gamma-rays). It crystallizes in a cubicgarnet known as YAG. In this complex crystal yttrium ions can besubstituted by rare earths leading to emit photons in a wide range ofwavelengths (UV-Vis-IR). In particular praseodymium replaces yttrium tooccupy octahedral sites of D₂ symmetry in YAG with a ground state is ³H4multiplet split by crystal field into 9 sublevels. 4f2 states of higherenergy give rise to spectrally narrow parity forbidden opticaltransitions in the visible and the IR. See FIG. 6.

In this investigation we have developed nanophosphors that efficientlyemit UV photons in the range of λ=300˜400 nm. This strong UV emission isefficiently activated with X-ray radiation (radioluminescence).

Experimental. Yttrium aluminum garnet doped with praseodymium(Y₃Al₅O₁₂:Pr or YAG-Pr) were obtained by the combustion synthesis method[Lopex, O. A., et al., 1997, J. Lum., 90:1]. Commercial precursors forthe synthesis were yttrium nitrate [Alfa Æesar Y(NO₃)₃.6H₂O 99.99%],aluminum nitrate [Alfa Aesar Al(NO₃)₃.6H₂O 99.99%], praseodymium nitrate[Alfa Aesar Pr(NO₃)₃.6H₂O 99.99%] and deionized water were pooledtogether in a reaction beaker. Carbohydrazide [CH₆N₄O, Alfa Aesar 98%]fuel was added and stirred for 20 min to form a homogenous solution. Thereaction vessel containing the mixture was transferred into a pre-heatedat ˜530° C. in order to ignite the reaction. After the reaction (3˜5min) a white foamy product is obtained which is finely crushed withmortar and pestle prior to post-annealing treatment at 1200° C. for 2hr. It has been found that heat treatments are crucial in order toenhance luminescence [Bosze, E. J., et al., 2007 J. Am. Ceram. Soc.,90:2484]. Additional details with a description of the reaction andprocedure for producing these phosphors are described in Bosze et al.2003 [Bosze, G. A., 2003, Mat. Sci. Eng., B97:265]

Typical X-ray diffraction (XRD) patterns of the powders were analyzedusing a Phillips X'pert diffractometer equipped with CuK_(α) radiation(λ=0.15406 nm). Measurements in a 2θ=15-80° range were taken with a stepsize of 0.02° and a 1 sec dwell per point. Transmission electronmicroscopy (TEM) images were obtained with a JEOL-2010 operated at 200kV accelerating voltage. Photoluminescence (PL) spectra were collectedwith a Hitachi spectofluorometer Model FL-7000. Radioluminescencemeasurements were performed under a x-Ray photons of 50 MeV. Allmeasurements were performed at room temperature.

Results. FIG. 7 shows a typical XRD pattern corresponding to thePr-doped Y₃Al₅O₁₂ nanocrystalline samples produced by combustionsynthesis and post-annealed at 1200° C. for 2 hr. This XRD pattern issimilar to that reported in the literature for yttrium aluminum garnetcrystalline structure. Identical XRD patterns were obtained for allpraseodymium concentrations used in the present investigation.

In FIG. 8 a representative TEM (for the same sample as in FIG. 7) of the1.0 at. % Pr-doped nanophosphor produced by combustion synthesis isshown. Morphology of this sample reveals a large number of irregularnanocrystallites of sizes in the range of 5.0-50.0 nm, with the mostfrequent size of around 30.0 nm, which confirms this phosphor powder asnanocrystalline material. Some porosity is observed which increases theamount of grain surface area which may cause the enhancement inluminescence intensity.

After ultrasonic processing for 10 min in isopropyl alcohol we havesucceeded in the deagglomeration of YAG-Pr clusters obtained bycombustion synthesis. See FIG. 9. These nanophosphor clusters areexploded by the ultrasonic vibrations at 24 MHz and single YAG:Prnanoparticles in the range of 10-40 nm or clusters with smaller size ofthe order of 40-70 nm.

Photoluminescence measurements were also performed for the YAG-Prnanophosphors. We found an optimum excitation wavelength at λ_(Exc)=292nm for all different concentrations of Pr. FIG. 10 illustrates thephotoluminescence characteristics for different concentrations of Pr inthe YAG host lattice. It can be observed that the maximum PL emissionoccurs in the sample doped with 1.0 at. % Pr the photoluminescence isoptimum. This UV emission contribution centered at λ=320 nm can beascribed to ⁵D₁→³P₂ and a much lower PL emission band due to ⁵D₁→³H₄electronic transitions inside the Pr³⁺ ion is also detected at λ=490 nm

As depicted in FIG. 11, radioluminescence spectra were investigated for(Y_(1-x)Pr_(x))₃Al₅O₁₂ with different Pr concentration of x=0.5, 1.0,1.5, 1.75 at. %. with X-ray excitation under 50 KeV.

Treatment of solid tumor. Recent reports by Munson et al., havedemonstrated accumulation of liposomes carrying Dox at rat C6 gliomabrain tumors with a striking suppression of tumor growth [Munson, J. M.et al. 2012, Sci Transl Med 4:127-136; Munson, J. et al. 2013, CellCycle 12:2200-2209]. This illustrates that liposomal agents canconcentrate at brain tumors via leaky vessels (enhanced permeability andretention, EPR-effect) and exert a useful therapeutic effect. We haveprepared liposomal Dox and demonstrated accumulation at tumors andeffective suppression of tumor growth (FIGS. 25A-25C). Our breast cancerbrain metastases model accurately emulates the human disease[Baschnagel, A. et al. Mol Cancer Ther 8:1589-1595].

Embodiments (Section IV)

Embodiments of the subject matter disclosed herein include EmbodimentsP1-P5 following.

Embodiment P1. A radiation activated nanoparticle assembly including amultilayered nanoparticle in external contact with a lipid-proreagent,the multilayered nanoparticle including a scintillator core, an inerthydrophilic shell, and an external lipid bilayer; the lipid-proreagentincluding a head group, a plurality of tail groups, and a proreagentattached at the head group with a photocleavable linker.

Embodiment P2. The radiation activated nanoparticle assembly accordingto embodiment P1, wherein the plurality of tail groups contact theexternal lipid bilayer.

Embodiment P3. The radiation activated nanoparticle assembly accordingto embodiment P1, wherein the proreagent is released from thelipid-proreagent by scission of the photocleavable linker.

Embodiment P4. The radiation activated nanoparticle assembly accordingto embodiment P1, wherein the scission results from release of lightfrom the scintillator core in response to radiation impinging thereon.

Embodiment P5. The radiation activated nanoparticle assembly accordingto embodiment P1, wherein the proreagent is a therapeutic agent, abiodistribution agent, or a labeling agent.

EXAMPLES

A major contribution disclosed herein is a platform (e.g., a compositiondisclosed herein) that releases an agent from a scintillator nanocrystalby low doses of radiation in place of light so that a photocleavablelinker is broken thereby releasing the agent. In this way drugs can beutilized as prodrugs and released. Indeed, radiation allows deeppenetration, e.g., in tissues which are not susceptible to UV-visiblelight exposure. The scintillator lights up in response to radiation,thereby breaking a chemical linker, and releases the agent. Theradiation can penetrate all tissues and many inert substancescompletely. Previous inventions have used toxic doses of radiationtogether with drugs to kill tumors. This results in considerable damageto the host, and limits the area that can be treated. In the approachdisclosed herein there is employed low doses (<1 Gy) of radiation athigh dose rate and pulsed, which serve to activate the chemical agent(e.g., prodrug). This means that a wide area and delicate anatomicalregions can be treated, and treated repeatedly over several weeks withminimal damage to normal tissues. This larger scale of treatmentincreases the probability that metastatic sites and new metastases willbe included in the treatment volume along with the primary tumor. Thenanoparticle prodrug platform when decorated with ligands or antibodiesfor cell surface receptors may facilitate entry into tumor cells andalso traversal of the blood brain barrier. In the latter case while theprodrug would be taken up elsewhere in the body, release of the activedrug would only occur in the brain after localized irradiation.Elsewhere the prodrug bound to the nanoscintillator composition wouldclear.

Example 1. (Lu_(1-α-β)Y_(α)Pr_(β))₂SiO₅ Powders with Fast Decay TimeObtained by the Combustion Synthesis Method

Abstract. UV-emitting (Lu_(1-α-β)Y_(α)Pr_(β))₂SiO₅ (0.1≤α≤0.4, β=0.05,0.005) nanophosphors were prepared by combustion synthesis andpost-annealed in air at 1200° C. at different annealing times.Structural and optical properties are investigated by X-Ray Diffraction,Raman Spectroscopy and Photoluminescence measurements. The formation ofmonoclinic (Lu,Y)₂SiO₅ solid solution was confirmed by X-ray diffractionas a majority phase. and under short-UV excitation the nanophosphorsyielded a strong UV-emission consisting of two bands with maximumemissions located at λ=280 nm and λ=315 nm, both corresponding to thePr³⁺ forbidden transitions ⁵D₂→4f (³P₂) and ⁵D₁→4f (³H₄), respectively.Furthermore, luminescence decay times of 10-16 ns were measured, whichdepend on the rare earth ion concentration.

Introduction. In the last decade a great effort has been dedicated tothe investigation of luminescent particles for multiple applicationsincluding novel types of flat TV's, electroluminescent panels, radiationdetectors and scintillator panels for X-Ray radiography among others. Inparticular. rare earth activated oxides, oxynitrides or silicates havebeen widely studied due to their d applications in the medical imagingfield as detectors (scintillators) in positron emission tomography (PET)equipment [H. Zaidi and A. Alavi. PET Clinics 2 (2) (2007) 109]. LSOpowders have been produced to substitute single crystals [D. W. Cooke,et al., J. Appl. Phys. 88 (12) (2000) 7360], and Lu has been replaced byY for the fabrication of (Lu,Y)₂SiO₅ (LYSO) crystals to reduce the highmanufacturing cost [H. Loudyi, et al., Opt. Mater. 30 (1) (2007) 26].There other reference of synthesized Ce-activated LYSO powders bycombustion synthesis method, also obtaining a solid solution of the twosilicates [M. Aburto-Crespo, et al., J. Lum. (2013) In Press]. BesidesCe³⁺, there are other rare earth ions that can be used as activator ionsin a scintillator material; one of them is Pr³⁺[C. W. E. Van Eijk, etal., IEEE Trans Nucl Sci. 41 (4) (1994) 738]. We here report a newalternative of scintillating materials: the synthesis of Pr³⁺-activatedLYSO powders using a low cost and fast method, combustion synthesis. Theinfluence of the Y/Lu ratio and Pr³⁺ concentration on the luminescenceproperties were examined. The combustion method is a fast and effectiveway to obtain micro or nano-scaled powders.

Experimental. The (Lu_(1-α-β)Y_(α)Pr_(β))₂SiO₅ powders were prepared bycombustion synthesis using lutetium nitrate [Alfa Aesar Lu(NO₃)₃.6H₂O98.%], yttrium nitrate [Alfa Aesar Y(NO₃)₃.6H₂O 99.9965%],nanocrystalline silicon oxide [SiO₂], praseodymium nitrate [Alfa AesarPr(NO₃)₃.6H₂O 99.99%] as the precursors, and hydrazine [Alfa Aesar N₂H₄98.5%] as the reductive fuel. α varied between 0.1-0.4 and β was either0.005 or 0.05 (see Table 1-I for sample identification and α and βvalues). The precursors were fully dissolved in 20 ml of deionized waterin a quartz beaker using a magnetic stirrer. After adding the hydrazine,a homogeneous solution was obtained with a gelatinous consistency. Thequartz beaker was introduced into the high-pressure stainless steellaboratory-built reactor. Next, the temperature was raised to 105° C.and a continuous argon flow was established in order to purge and createan inert atmosphere. The reactor exhaust valve was closed and the argoninlet opened to pressurize the reactor (3.45 MPa). Using a higherpressure than ambient, nanoparticles are obtained [C. E.Rodriguez-Garcia, et al., J. Phys. D: Appl. Phys. 41 (9) (2008) 092005]and some evidence indicates that a smaller particle size increases theluminescence emission of the phosphor [M. Nikl, et al., IEEE Trans NuclSci. 55 (3) (2008) 1035]. Once the reaction was completed, the exhaustvalve was opened to release the pressure and the argon was used to flushthe reactor until it cooled down to 40° C. The same procedure wasrepeated for all samples. All powders were thermally treated in air at1200° C. for different periods of time ranging from 2-4h. Table 3 liststhe annealing time for each sample.

TABLE 3 Concentrations of Y³⁺ and Pr³⁺, post-synthesis annealing timeand photoluminescence decay time for combustion synthesized(Lu_(1−α−β)Y_(α)Pr_(β))₂SiO₅ powders. All samples were thermally treatedin air at 1200° C. Annealing time Decay time x y (h) (ns) LYSOP1 0.20.05 2 16 LYSOP2 0.2 0.05 4 11 LYSOP3 0.49 0.005 4 10

Powder diffraction patterns were obtained over the scattering range2θ=10-60° with steps of 0.02° using Cu Kα radiation (λ=0.15406 nm) in aPhilips X'pert diffractometer. Photoluminescence (PL) spectra werecollected at room temperature with a Hitachi FL-4500 fluorescencespectrophotometer with excitation and emission slit of 2.5 nm andwavelength scan speed of 1200 nm/min. The spectrofluorometer uses a 150W Xe lamp for sample excitation. The Pr³⁺ decay emissions curves wereanalyzed with a HORIBA Jobin Yvon SPEX spectrofluorometer with aspectral resolution of 0.25 nm, exciting the samples with aFluoroLog—TCSPC pulse laser-diode, which produces pulses between 100-200ps with broadband output from 180-780 nm. All the scintillatingmeasurements were also performed at room temperature.

Results and Discussion. FIG. 12 shows the powder XRD patterns. Formationof a (Lu,Y)₂SiO₅ solid solution was obtained as a majority phaseregardless of the composition and post-synthesis annealing time. Thediffraction peaks of the (Lu,Y)₂SiO₅ powders match perfectly with thereported monoclinic Lu₂SiO₅ (JCPDS 41-0239). Small traces of tworesidual phases were identified as Lu₂Si₂O₇ and Lu₂O₃. This is in goodagreement with XRD data reported by other groups for this compound [H.Loudyi, et al., Opt. Mater. 30 (1) (2007) 26], which confirms theformation of an LYSO solid solution. It is important to note that no Proxide phase was detected—a clear indication of excellent Pr³⁺incorporation into the host lattice.

TEM micrographs (FIG. 13) show the morphology of sample LYSOP1. FIG. 13(left panel 1) reveals rough surfaced rods of approximately 0.6 μmlength. FIG. 13 (right upper panel 2) shows bars with clearlyhemispherical endings of about 70 nm diameter. FIG. 13 (lower panel 3)depicts planes of a crystal with interplanar spacing of 0.350 nm,associated to the (2 0 2) planes.

Photoluminescence (PL) spectra were collected at room temperature withshort UV-excitation (λ_(ex)=250 nm). All samples emitted a visible dimred light. FIG. 14 shows the excitation and emission spectra of allLYSOP samples. The emission spectra range from 257 nm to 350 nm, withtwo major peaks centered at λ=280 nm and λ=315 nm. Pr³⁺-activated LSOsamples with emission spectra from 270 nm to 370 nm have been reported[D. W. Cook, et al., Opt. Mate. 27 (12) (2007) 1781] which correspond to4f→4f transitions. It should be emphasized that when Pr³⁺ ions are on amoderately strong crystalline field lattice (˜6 eV) it is possible toobserve the 5d→4f transitions. For the LYSO host materials in this studythe band gap was 5.81 eV. On the other hand, the 5d→4f transitions arecaused when a rare earth ion is in presence of an electrical field. Onthe LYSO host lattice, when a rare earth ion replaces a Lu ion, the unitcell has the form RE₂(SiO₄)O, (RE=rare earth) [P. C. Ricci, et al., J.Raman Spectrosc. 39 (9) (2008) 1268]. In this study, the negative chargeof the silicate ion in the unit cell acts as an electrical field aroundthe Pr³⁺ ion consequently, the ¹S₀ level moves above the ⁵D₁ level (FIG.15); a second mechanism that makes the scintillating 5d→4f transitionspossible. All spectra show ⁵D₂→4f (³P₂) and ⁵D₁→4 f (³H₄) transitions.The spectrum matches the data reported for LSO scintillator crystalsactivated with Pr³⁺[M. Nikl, et al., 2005. Chemical Physics Letters. 410(4-6) (2005) 218].

The reported scintillating decay time for LYSO single crystals is of theorder of 40 ns [A. J. Wojtowicz, et al., Opt. Mater., 28 (1-2) (2006)85]. The luminescence lifetime of a phosphor follows an exponentialdecay [Joseph R. Lakowicz. Principles of Flourescence Spectroscopy,Springer, New York, USA, (1983)], which is represented by the followingformula:I=I _(o) exp^(−kt)where I is the luminescence emission intensity, I_(o) is the initialintensity, k is the relaxation rate and t is the decay time [Joseph R.Lakowicz. 1983, Id.]. Room temperature luminescence decay time profilesof the Pr-doped emission monitored at 280 nm upon excitation at 252 nmvaried between 10-16 ns as shown in FIGS. 16A-16B. FIG. 16A shows theLYSOP1 sample decay curve corresponding to the emission peak located at280 nm (FIG. 14). In this case, the decay follows two exponentialcurves. We assume that the shortest time for sample LYSOP, is due to ahost lattice emission, independent of the activator. FIG. 16B shows thedecay curves of samples LYSOP2 and LYSOP3. Both decay curves were fittedonly to one exponential curve, so we assume that there is no presence ofemission by the host lattice in none of the two samples. The measurementcorresponds to the peak located at 275 nm (LYSOP2 sample) and 277 nm(LYSOP3 sample) shown in FIG. 14. Table 3 summarizes the calculateddecay times for the set of samples. PET detectors have to work at highcount-rates, so it is important that the decay time of the scintillatorshould be short (less than 40 ns) [C. Ronda, et al., Mater. Res. Soc.Symp. Proc. 1111 (2009) 1111-D08-01]. Decay time of LYSO single crystalscan be modified by varying the Y, for higher Y concentrations,scintillating times are longer, but when the concentrations vary between5-10%, there is no difference in the decay time [M. Nikl, et al., IEEETrans Nucl Sci. 55 (3) (2008) 1035]. In this study, the sample with thelargest concentration of Y and the smaller Pr³⁺ concentration (sampleLYSOP3) yields the shortest decay time. The resulting decay time valuesin this study contrast with the previously decay time values reportedfor Ce-activated LYSO singles crystals [M. Nikl, et al., IEEE Trans NuclSci. 55 (3) (2008) 1035], Ce-activated LYSO powders [C. W. E. Van Eijk,et al., 1994, Id.] or even Pr-activated LSO single crystals [M. Nikl, etal., Journal of Crystal Growth. 292 (2) (2006) 416], but a detailedexperiment must be performed in order to clarify the(Lu_(1-α-β)Y_(α)Pr_(β))₂SiO₅ materials behavior.

Conclusion. UV emitting (Lu_(1-α-β)Y_(α)Pr_(β))₂SiO₅ (0.1≤α≤0.4, β=0.05,0.005) powders were successfully synthesized by the combustion synthesismethod. The Pr³⁺ was well incorporated into the (Lu,Y)₂SiO₅ host latticeas confirmed by X-ray diffraction analysis. Transmission electronmicroscopy revealed the formation of bar-type crystals of 0.6 μm length.Photoluminescence measurements (λ_(exc)=250 nm) showed a very brightUV-emission composed of two wide peaks with maximum emissions centeredat λ=280 nm and λ=315 nm, both corresponding to the Pr³⁺ forbiddentransitions ⁵D₂→4f (³P₂) and ⁵D₁→4 f (³H₄), respectively. Moreover,these powders produce luminescence decay times in the range of 10-16 ns,with the shortest decay time for the sample with the smallest Pr³⁺concentration. The application of (Lu_(1-α-β)Y_(α)Pr_(β))₂SiO₅ powdersas suitable PET components is not verified yet. In future investigationswill be necessary to make a radioluminescence analysis to observe theirbehavior under high-energy excitation.

Example 2. Thermoluminescence and Afterglow Low Dose β Dosimetry Basedon Nanocrystalline YAG-Pr³⁺ Produced by Combustion Synthesis

Abstract. In this work thermoluminescence (TL) and afterglow (AG)properties of Pr-doped Y₃Al₅O₁₂ (YAG-Pr³⁺) nanocrystalline phosphors(30.0 nm) produced by combustion synthesis method were investigated fordosimetric and medical applications. The TL and AG results suggest thatPr³⁺-doped Y₃Al₅O₁₂ (YAG-Pr³⁺) nanocrystalline phosphor is an excellentcandidate for β irradiation medical dosimeter applications in the rangeof 0-20 Gy. Both thermoluminescence and afterglow properties wereoptimal with 0.5% Pr⁺³. Following irradiation AG and TL dosimetry wereperformed for 0-5 hrs, and more than 5 hrs, respectively. Fluorescencewas linear for radiation doses of 0-20 Gy, with no apparent saturationup to 200 Gy.

Introduction. Materials exhibiting thermoluminescence (TL) and afterglow(AG) properties in response to ionizing radiation are very important asdetectors in dosimetric and potentially other medical applications suchas photodynamic therapy [Azorin, J., et al., Phys. Stat. Sol. A 138(1993) 9-46; Bos, A. J., Radiat. Meas., 33 (2001) 737-744; Bos, A. J.,Radiat. Meas., 41 (2006) S45-S56]. TL dosimetric materials shouldpossess a wide linearity range with absorbed dose, high sensitivity, lowfading, radioresistance, and they should be chemically inert [McKeever,S. W. S., et al., 1995, Thermoluminescence Dosimetry Materials:Properties and Uses, Nuclear Technology Publishing, Ashford, UK]. In thelast few years the search for improved thermoluminscent materials hasresulted in the characterization of various crystalline oxide rare earthactivated materials. Promising candidates include Lu₂SiO₅ (LSO) andY₂SiO₅ [Zorenko, Y., et al., Opt. Mater., 34 (2012) 1969-1974.],lutetium aluminate perovskite LuAlO₃, lutetium yttrium orthosilicate(LYSO) and yttrium aluminum garnet (YAG) [Wojtowicz, A. J., et al., J.,Opt. Mater., 28 (2006) 85-93], Interesting luminescence properties havebeen reported for nanophosphor hosts activated with rare earth ionsnotably of the garnet group [Speghini, A., et al., Opt. Mater., 33(2011) 247-257]. The garnet group has the general formula of X₃Y₂(SiO₄)₃where the X site is usually occupied by divalent cations (Ca²⁺, Mg²⁺,Fe²⁺) and the Y site by trivalent cations (Al³⁺, Fe³⁺, Cr³⁺, Pr³⁺) in anoctahedral/tetrahedral frame with (SiO₄)₄ occupying the tetrahedral. Thebest garnet candidate for luminescent material is YAG, which providesunique optical properties when doped with trivalent lanthanide ions[Speghini, A., et al., 2011, Id.]. These Y₃Al₅O₁₂ (YAG) oxyorthosilicatematerials in nanocrystalline form have been studied forthermoluminescent dosimetry (TLD) of ionizing radiation [Zhydachevskii,Ya., et al., Radiat. Meas., 45 (2010) 516-518; Zhydachevskii, Y., etal., Radiat. Meas., 46 (2011) 494-497; Jayaramaiah, J. R., et al.,Mater. Chem. Phys., 130 (2011) 175-178; Premkumar, H. B., et al.,Spectrochim. Acta, Part A. 96 (2012) 154-162] because they are excellenthosts with high thermal stability [Antic-Fidancev, E., et al., AlloysCompd., 341 (2002). 82-86], high effective atomic number (Z_(eff)=31.4),high sensitivity and linear response in the dose range 1×10⁻⁴ to 1×10³Gy [Zhydachevskii, A. et al., Radiat. Meas., 42 (2007) 625-627].Praseodymium (Pr³⁺) impurities in yttrium-based compounds areparticularly interesting as Pr³⁺ ions occupy several different sites inthe complex yttrium dodecahedral lattice [Gruber, J. B., et al., No.HDL-TR-2171, Harry Diamond Labs Adelphi, Md. (1989)].

Recently, luminescent properties of 58 nm YAG-Pr³⁺ crystals werereported together with a model explaining the differences between nanoand crystalline forms of YAG-Pr³⁺[Ozen, G., et al., 2013, Id.].Moreover, R. A. Rodriguez et al., studied the thermoluminescence (TL)and luminescence of YAG: Er³⁺,Yb³⁺ under high β radiation doses, andfound that the introduction of impurities modifies the crystal structureand therefore localized trapping states [Rodriguez, R. A., et al., Opt.Mater., 27 (2004) 293-299]. However, a mechanism for explainingluminescence relaxation is still needed, and accordingly, Yukihara andco-workers [Yukihara, E. G., et al., J. Lumin., 133 (2013) 203-210] haveproposed a phenomenological model of energy levels for luminescentrecombination to explain thermometry properties of YAG-Pr³⁺.Nonetheless, dosimetric properties for low dose radiation and formedical applications, particularly TL and AG, of YAG-Pr³⁺ nanopowderremained to be addressed both experimentally and theoretically.

Here we analyzed TL and AG dosimetric properties of Pr-doped YAGnanophosphers synthesized by the combustion method at with Pr³⁺ at 0.5,1.0, 1.5 and 2.0%. In the context of both TL and AG applications, wefound that 0.5% Pr³⁺ doping concentration provided the best combinationof sensitivity, thermoluminscence output, and afterglow properties. Thepresent data support further exploration of YAG nanophosphors fordosimetric and nanomedical applications.

Experimental

Synthesis of YAG-Pr³⁺ by combustion method. Nanocrystalline phosphors ofPr-doped Y₃Al₅O₁₂ (YAG-Pr³⁺) were produced by combustion synthesis[Lopez, 0. A., et al., J. Lumin., 71 (1997) 1-12]. The startingmaterials for the synthesis were aluminum nitrate [Alfa ÆesarAl(NO₃)₃.6H₂O 99.99%], yttrium nitrate [Alfa Æesar Y(NO₃)₃.6H₂O 99.99%]and praseodymium nitrate [Alfa Æesar Pr(NO₃)₃.6H₂O 99.99%] as precursorsthat were dissolved in 30 ml of deionized water in a quartz beaker, andthen carbohydrazide [CH₆N40, Alfa Æesar 98%] was added as a reductivefuel forming a homogeneous solution. The chemical precursors wereadjusted in order to obtain (Y_(1-x)Pr_(x))₃Al₅O₁₂ with different Pr³⁺concentration of x=0.5, 1.0, 1.5 or 2.0 at. %. Quartz beakers containingmixtures with different Pr³⁺ concentrations were individually placedinto a pre-heated oven (˜530° C.) in order to induce the reaction. After3-5 minutes the reaction takes place and a white foamy product isobtained which is finely crushed with mortar and pestle prior topost-annealing treatment at 1200° C. for 2 hours. It has been found thatheat treatments are crucial in order to enhance luminescence. Additionaldetails with a description of the reaction and procedure for producingthese phosphors are described in Bosze et al. [Bosze, E. J., et al.,Mater. Sci. Eng. A, 97 (2003) 265-274; Bosze, E. J., et al., J. Am.Ceram. Soc., 90 (2007) 2484-2488].

Characterization. X-ray diffraction (XRD) analysis of the powders wereperformed in a Phillips X'Pert diffractometer equipped with CuK_(α)radiation (λ=0.15406 nm). Measurements in a 2θ=15-80° range were takenwith a step size of 0.02° and a 1 sec dwell per point. Transmissionelectron microscopy (TEM) images were obtained with a JEOL-2010 operatedat 200 kV accelerating voltage. Photoluminescence (PL) spectra werecollected with a fluorescence spectrophotometer (Hitachi FL-4500). Allmeasurements were performed at room temperature (RT).

Thermoluminescence and afterglow measurements. The TL and AG experimentsand β-irradiation (Sr-90, 40 mCi, 0.1 Gy/s in quartz) were performed ina Risø TL/OSL reader (model TL/OSL DA-20). The AG decay curves wererecorded at RT, the heating rate of the TL readouts was 5 K/s, in thetemperature range from RT to 350° C. The pellets of 5 mm diameter, 1 mmof thickness and 100 mg used in AG and TL experiments were prepared withpowder and exposed to p irradiation.

Results and Discussion.

X-Ray Powder Diffraction. FIG. 7 shows a typical XRD patterncorresponding to the Pr³⁺ doped Y₃Al₅O₁₂ nanocrystalline samplesproduced by combustion synthesis and post-annealed at 1200° C. for 2 hr.This XRD pattern is similar to that reported in the literature for theYAG crystalline structure [Hess, N. J., et al., J. Mater. Sci., 29(1994) 1873-1878]. Similar XRD patterns were obtained for allpraseodymium concentrations used in the present investigation.

Crystal structure of YAG-Pr³⁺. In FIG. 8, a representative TEM (samesample as in FIG. 7) of the 1.0 at. % Pr³⁺-doped nanophosphor is shown.Morphology of this sample reveals a large number of irregularnanocrystallites of sizes in the range of 5.0-50.0 nm, with the mostfrequent particle size of around 30.0 nm, which confirms this phosphorpowder as nanocrystalline material. Some porosity is observed whichincreases the amount of grain surface area, which may be related to theenhancement in luminescence intensity.

Photoluminescence property. Photoluminescence measurements were alsoperformed for the YAG-Pr³⁺ nanophosphors. We found an optimum excitationwavelength at λ_(Exc)=292 nm for all different concentrations of Pr³⁺.FIG. 10 illustrates the photoluminescence characteristics for threedifferent concentrations of Pr³⁺ in the YAG host lattice. It can beobserved that the maximum PL emission occurs in the sample doped with1.0 at. % Pr³⁺. This UV emission centered at λ=320 nm can be ascribed to⁵D₁→³P₂ transition, and a much lower PL contribution due to ⁵D₁→³H₄electronic transitions within the praseodymium ion is also detected atλ=490 nm [Kolesov R., et al., Nat. Comm., 3 (2012) 1029-1036].

Thermoluminescence (TL) measurements. The TL fading for YAGnanophosphors with 0.5, 1.0, 1.5 and 2.0 at. % concentrations of Pr³⁺after 1, 2, 5, 10 and 24 hours are shown in FIGS. 17A-17D, respectively.For all samples, TL intensity is inversely proportional to Pr³⁺concentration. TL glow curves in YAG-Pr³⁺ consist of three broad peaksat approximately 200, 275 and 375° C. It is observed that the 200° C.peak decreases significantly with time whereas the peak at 375° C.remains more stable. This behavior may be ascribed to shallow trapsgenerated by spurious contaminants and high frequency factors forreleasing electrons from these traps. In general, thermoluminescent glowcurves are composed for both different trapping states and depthscorresponding to Pr³⁺ dopant defect centers with different structureposition, created during the ionizing radiation stage. In this case, TLfading behavior is mainly due to the higher temperature (375° C.) peak.At this point, it is important to note that similar results consistingin four peaks at 170, 210, 256 and 332° C. (main peak), were reported bythe deconvolution of the TL glow signal in nanocrystalline YAG by R. A.Rodriguez and collaborators [Rodriguez, R. A., et al., J. Phys. D: Appl.Phys, 38 (2005) 3854], where they related this TL behavior with thepresence of stronger major contents of defects in comparison with singleYAG.

As discussed by E. D. Milliken and collaborators [Milliken, E. D., etal., 2012, J. Lumin., 132: 2495-2504]. trivalent lanthanides like Pr³⁺are likely to act as hole traps that are stable at room temperaturebecause their electronic levels are well above the top of the valenceband (2.06 eV). Such defects can capture a hole from the valence band:h++Pr³⁺→Pr⁴⁺ 1.

If the trapped hole subsequently captures an electron from theconduction band, emission from this cation will be observed:e−+Pr³⁺→(Pr³⁺)*+hν 2.

They found that the excitation band of the transition 4f ground state to5d2 level of YAG-Pr³+ present peaks of energy estimated to 2.06 eV asthe energy difference between Pr³⁺ ground state and the top of thevalence band [Milliken, E. D., et al., 2012, Id.].

The Pr³⁺ concentration response of β irradiated (20 Gy) YAG samples wasinvestigated by plotting the integrated TL signal as function of timeafter exposure and is shown in FIG. 18. According to this, we have foundthat TL of YAG-Pr³⁺ nanophosphors is stable after five hours for allPr³⁺ concentrations. Moreover, it can be observed that the strongestfading signal is associated with 0.5 at. % Pr³⁺ and, as describedbefore, TL intensity decreases as Pr³⁺ concentration increases.

For medical applications we found that YAG-Pr³⁺ showed strong linear TLintensity behavior with doses of β irradiation up to 20 Gy. No apparentTL saturation was observed for 200 Gy but a superlinear response at highdoses for all samples. Also, we confirmed that 0.5 at. % Pr³⁺ is mostintense for all doses, see FIG. 19. This linear response and low fadingmakes YAG-Pr³⁺ a good candidate for TL dosimetry.

Afterglow (AG) Curves. An alternative form to achieve β dosesimmediately after irradiating tissues in medicine is known as AG decay.In FIGS. 20A-20D the AG decay curves of YAG nanophosphor with fourdifferent concentration of Pr³⁺ as a function of the β irradiation doseare compared. This AG corresponds to samples irradiated at 1, 2, 5, 10,20, 50, 100, 200 and 500 Gy. For all the samples, it can be observedthat intensity increases as the dose increases, as observed in TLbehavior. However, opposite to the TL, no saturation was found even tohigh doses of 500 Gy.

Also, YAG-Pr³⁺ 0.5 at. % has the best linear response of AG intensity vsdose, and it is possible to detect low doses as 20 Gy (FIG. 21). It isimportant to note that although YAG-Pr³+ 2.0 at. % shows the lowest AGresponse, this sample depicted the highest linearity range, up to 200Gy.

Conclusion. We found that combustion synthesis produces nanocrystallineYAG-Pr³⁺ in the 5-50 nm diameter range. Pr³⁺ is incorporatedsubstitutionally in Y garnet positions as confirmed by XRD and TEMmeasurements. YAG-Pr³⁺ exhibits an intense TL and AG signal, which isuseful for TL-based dosimetry to achieve retrospective doses and for AGdosimetry to determine real time doses. Our results show that samples ofYAG-Pr³⁺ incorporating 0.5% of Pr³⁺ during crystal growth are the mostoptimal for both TL and AG dosimetric approaches. The results supportfurther investigation of YAG-Pr³⁺ nanocrystals for dosimetric andbiomedical applications.

Example 3. Photo Emission Properties of Doped Nanocrystals

Photon emission per MeV. The radioluminescence emission properties ofthe nanoscintillator crystals tabulated in Table 4 following wereassayed using methods known in the art. See e.g., Jung, J. Y., et al.,J. Luminescence 2014, 154:569-577. The term “Act.” refers to“activator.”

TABLE 4 Photon emission per MeV of X-rays and emission peakwavelength(s) Emission Emission Luminosity peak peak Composition Act.(photons/MeV) (nm) 1 (nm) 2 GdOCl:Ce Ce³⁺ 75% of ZnS:Ag 365 410 YOCl:CeCe³⁺ 52% of ZnS:Ag 365 405 LaBr₃:Cc Ce³⁺ 74,000 375 CeBr3 Ce³⁺ 68,000371 LaBr₃:Cc Ce³⁺ 61,000 356 387 K₂LaCl₅:Ce Ce³⁺ 49,300 347 372 LaCl3:CeCe³⁺ 48,000 350 CeCl3 Ce³⁺ 46,000 350 K2LaBr5:Ce Ce³⁺ 40,000 359 391NaGdCl4:Ce Ce³⁺ 39,400 350 370 GdCl3:Ce Ce³⁺ 38,000 350 370 BaGdCl5:CeCe³⁺ 35,000 363 389 Gd2Si2O7:Ce (GPS) Ce³⁺ 35,000 372 394 Rb2LiLaBr6:CeCe³⁺ 33,000 363 387 Ba2GdCl7:Ce Ce³⁺ 30,000 355 227 K2CeCl5 Ce³⁺ 30,000370 Lu2Si2O7:Ce (LPS) Ce³⁺ 26,000 378 Cs2NaCeBr6 Ce³⁺ 25,000 377 400Rb2LiCeCl6 Ce³⁺ 23,100 370 Lu2Si2O7:Ce (LPS) Ce³⁺ 23,000 380Cs2LiYCl6:Ce (CLYC) Ce³⁺ 21,600 370 PrBr3:Ce Ce³⁺ 21,000 365 395LuAlO3:Ce Ce³⁺ 20,500 365 YAlO3:Ce (YAP) Ce³⁺ 20,100 365 Cs2LiYCl6:CeCe³⁺ 18,400 372 400 LuPO4:Ce Ce³⁺ 17,200 360 CeCl3(CH3OH)4 Ce³⁺ 16,600364 YAlO3:Ce (YAP) Ce³⁺ 16,200 347 Y2O3 SX 15,480 370 LuAlO3:Ce Ce³⁺11,400 365 BaBr2:Ce Ce³⁺ 10,300 370 370 KYP2O7:Ce Ce³⁺ 10,000 380 Y2O3SA 9,300 350 Y₃Al5O12:Pr Pr³⁺ 8,000 350 Sc2O3 SA 7,700 350 Cs2LiLuCl6:CeCe³⁺ 7,000 370 410 Li3YCl6:Ce Ce³⁺ 6185 360 385 NaLuP2O7:Ce Ce³⁺ 6,000370 LuOCl:Ce Ce³⁺ 5,500 380 BaCl2:Ce Ce³⁺ 5,200 349 373 LuScBO3:Ce Ce³⁺4,200 370 YOCl:Ce Ce³⁺ 3,500 380 CeBr3 + 1− Ce³⁺ 3218 365 Ca5(PO4)3F:CeCe³⁺ 3,200 354 412 CeBr3 + tert-butanol Ce³⁺ 3095 360 BaF2:Ce Ce³⁺ 2,200360 CeBr³⁺isobutanol Ce³⁺ 1920 360 BaP2O6:Eu Eu²⁺ 1,900 380 470 LaBO3:CeCe³⁺ 600 355 380 Ba5(PO4)3F:Ce Ce³⁺ 400 358 HfF4 SA 300 350 BaF2:Ce Ce³⁺350 LiYSiO4:Bi Bi³⁺ 350 LaBO3:Bi Bi³⁺ 365

Integrated intensity (350-370 nm). The integrated light emission ofnanoscintillators that emit in the range 300-400 nm was determined inresponse to radiation exposure (50 KeV X-rays, short pulse). The resultsdepicted in FIG. 22 demonstrate a significant increase in integratedintensity of YAG-Pr nanoscintillators relative to other indicatedsamples.

Peak intensity versus emission wavelength. Peak intensity versuswavelength in the range 250-750 nm is depicted in FIG. 23 for theindicated nanoscintillators. See also FIG. 11.

Example 4. Release of Chemical Agent

Release of chemical agent, e.g., doxorubicin, can be monitored duringdialysis of nanocrystals attached to the chemical agent by methods wellknown in the art, including e.g., absorption spectroscopy. Doxorubicinwas attached to a nanoscintillator (yttrium aluminum crystal doped withpraseodymium, YAG-Pr) via a photocleavable linker disclosed herein,which in turn was attached to streptavidin which coated the nanocrystal.Release of doxorubicin was assayed by absorption spectroscopy before andduring dialysis as a function of radiation exposure. As depicted in FIG.24A, at 1-hr after control or radiation exposure (2 Gy or 4 Gy),doxorubicin cleaves from the nanocrystal. The degree of release ofdoxorubicin correlates with radiation exposure. As depicted in FIG. 24B,after 24-hr dialysis, there is significant cleavage releasingdoxorubicin into the dialysis media.

Example 5. Sol-Gel Synthesis of YAG-Pr

Yttrium aluminum garnet (Y₃Al₅O₁₂, YAG) doped with praseodymium wassynthesized via the sol-gel method. Yttrium acetylacetonate(Y(C₅H₇O₂)₃), aluminum acetylacetonate (Al(C₅H₇O₂)₃), and praseodymiumnitrate (Pr(NO₃)₃) were used as precursors. The precursors were mixedwith the stoichiometry of Y:Al:Pr=3−x:5:x (x=0.01). Water was slowlyadded and mixed on a magnetic stirring hot plate at 80° C. until a gelformed. The gel was then calcinated at 700° C. for 3 hours, resulting inthe YAG-Pr powder. Previous investigations on sol-gel synthetic methodsmay be found in Puzyrev, I. S., et al., 2012, Glass Physics andChemistry, 38:427-430. Exemplary methods of sol-gel combustion synthesisof nanocrystalline YAG powers from metal-organic precursors arediscussed in Chen, D., et al., 2008, J. Am. Ceram. Soc., 91:2759-2762.

Example 6. Additional Embodiments

Applications of the compositions and methods disclosed herein have greatpotential in terms of the treatment of cancer, infectious diseases, andindustrial applications. For example, a radiation activated prodrug ableto significantly improve the management of solid invasive tumors ortreat infections with a minimum of side effects would be a highlyuseful. Another application includes using the disclosed compositions toactivate a series of particles carrying different reagents to carry outchemical reactions in machinery or underground pipes or tanks that canbe irradiated once enough particles have accumulated. This method couldbe used to repair or enhance the properties of machinery.

Moreover, the radiation activated prodrug strategy may be useful for arange of solid or diffuse tumors in addition to brain cancer. Thenanoparticle prodrug platform when decorated with ligands or antibodiesfor cell surface receptors may facilitate entry into tumor cells andalso traversal of the blood brain barrier. In the latter case while theprodrug would be taken up elsewhere in the body, release of the activedrug would only occur in the brain after localized irradiation.Elsewhere the nanoparticle prodrug would clear.

The disclosed methodology is also useful to concentrate antibiotics, inthe form of prodrugs, targeted to areas of inflammation/infection. Avery low level of localized radiation would be used to liberate thedrug(s) creating high local concentrations to overcome bacterialresistance just at the infection site, but the total body dose of theantibiotic would remain low thereby avoiding side effects.

This method may be useful for repairing tubing and conduits. Variouscomponents of a cement would be carried to and accumulate at sitesneeding repair, possibly via an externally applied magnetic field, andlocal, shaped irradiation would trigger release of cement components infissures or breaks. This may be particularly useful in corn silos forexample, as their internal workings are subject to cracking and they aredifficult and costly to repair.

Example 7. Design Considerations

There are disclosed a variety of nanoscintillator compositions usefulfor the methods disclosed herein. The yttrium-aluminum crystal dopedwith praseodymium (YAG-Pr) are observed to much more efficientlygenerate photons in response to radiation than did the barium germaniumoxide (BGO) scintillator, 4.5×10⁸ versus 1.5×10⁷, respectively (seee.g., FIG. 22). It is noted that both yttrium and praseodymium innon-soluble form are regarded as essentially non-toxic and yttrium isalready used for clinical applications. The YAG-Pr efficiently emittedphotons with KeV energy levels of x-rays, to the same extent as would bepredicted for MeV levels. Without wishing to be bound by theory, it isbelieved that this is very positive as it means that low and lessdamaging energy levels of radiation may be used to activate chemicalagents at a desirable site, e.g., anticancer prodrugs (or antibiotics)at tumors, thereby very much sparing normal host tissues. As proof ofconcept, we attached Dox to the scintillator crystal via aphotocleavable linker using streptavidin and were able to obtain astepwise release of Doc with escalating doses of irradiation using 1.1MeV gamma rays.

The effectiveness of the nanoscintillator synthesis was determined intwo ways. First, using electron microscopy it was demonstrated thatparticle shape and size, (i.e., 50-100 nm diameter and spherical) wereconsistent with shape and size criteria. Second, without wishing to bebound by theory, it is believed that the capacity for robust photonemission at the desired wavelength range of 350-470 nm with radiationexposure is acritical criterion of success. This was measured as thephotons per MeV ranged from zero to 74,000. Indeed, the YAG-Pr crystalshad the highest integrated emission between 350-370 nm (4.5×10⁸photons). This wavelength range is useful to break the photosensitivelinker (i.e., photocleavable linker) attaching a chemical agent moiety(e.g., doxorubicin therapeutic) to the nanocrystal scintillator. Againwithout wishing to be bound by theory, it is believed that scintillatorwavelengths longer than about 365 nm may not be energetic enough tobreak the linker, but could additionally interact with the chemicalagent moiety. Moreover, wavelengths shorter than about 365 may damagebiological molecules and the chemical agent.

Summary We investigated a variety of potential scintillator elements andcombinations for efficiency with low doses of radiation. We exploredmethods of synthesizing the best candidate compositions and the mostefficient crystals, and identified the most effective method. Wesystematically varied synthesis conditions, the percentage of doped, thetime of combustion, specialized heating, and the time of ultrasonicationto attain the most efficient scintillator crystals. We determinedoptimum conditions for a sophisticated and successful synthetic regime.We showed that our crystals when irradiated with x-rays robustly emitlight of a wavelength that will break a UV photosensitive linker. Thesecrystals which are non-toxic, have a composition which allow coating andattaching of a photocleavable linker with drug. We showed that lowenergy X-rays, in the KeV range, will cause scintillator nanocrystals toscintillate such that the emitted photons can break the photocleavablelinker and release a chemical agent moiety.

Embodiments

Further embodiments include the Embodiments 1-38 following.

Embodiment 1. A composition comprising a scintillator nanocrystal linkedto a chemical agent moiety through a scintillator-activatedphotocleavable linker.

Embodiment 2. The composition of embodiment 1, wherein said scintillatornanocrystal comprises a plurality of scintillator activators dispersedwithin a host crystal lattice.

Embodiment 3. The composition of embodiment 2, wherein said plurality ofscintillator activators are cesium, europium or praseodymium.

Embodiment 4. The composition of embodiment 2, wherein said plurality ofscintillator activators are cesium or praseodymium.

Embodiment 5. The composition of embodiment 2, wherein said plurality ofscintillator activators are praseodymium.

Embodiment 6. The composition of one of embodiments 1 to 5, wherein saidhost crystal lattice is a chloride host crystal lattice, bromide hostcrystal lattice, oxide host crystal lattice, iodide host crystal latticeor silicate host crystal lattice.

Embodiment 7. The composition of embodiment 6, wherein said host crystallattice is a lanthium bromide host crystal lattice.

Embodiment 8. The composition of embodiment 6, wherein said host crystallattice is an oxide host crystal lattice or silicate host crystallattice.

Embodiment 9. The composition of one of embodiments 1 to 5, wherein saidhost crystal lattice is a garnet host crystal lattice.

Embodiment 10. The composition of one of embodiments 1 to 5 or 9,wherein said host crystal lattice is an yttrium aluminum oxide hostcrystal lattice or gadolinium/yttrium aluminum oxide or a yttriumgallium/aluminum oxide host crystal lattice.

Embodiment 11. The composition of embodiment 1, wherein saidscintillator nanocrystal has the formula (Y_(1-x)Pr_(x))₃Al₅O₁₂, whereinx is 0.0075, 0.01, 0.0125, 0.015 or 0.0175.

Embodiment 12. The composition of one of embodiments 1 to 11, whereinsaid scintillator nanocrystal has a diameter from 25 nm to 300 nm.

Embodiment 13. The composition of one of embodiments 1 to 11, whereinsaid scintillator nanocrystal has a diameter from 50 nm to 250 nm.

Embodiment 14. The composition of one of embodiments 1 to 11, whereinsaid scintillator nanocrystal has a diameter from 50 nm to 200 nm.

Embodiment 15. The composition of one of embodiments 1 to 11, whereinsaid scintillator nanocrystal has a diameter from 50 nm to 150 nm.

Embodiment 16. The composition of one of embodiments 1 to 11, whereinsaid scintillator nanocrystal has a diameter from 50 nm to 100 nm.

Embodiment 17. The composition of one of embodiments 1 to 11, whereinsaid scintillator nanocrystal has a diameter of less than about 200 nm.

Embodiment 18. The composition of one of embodiments 1 to 17, whereinsaid scintillator nanocrystal emits photon emission peaks within 300 nmto 470 nm.

Embodiment 19. The composition of one of embodiments 1 to 17, whereinsaid scintillator nanocrystal emits photon emission peaks within 350 nmto 470 nm.

Embodiment 20. The composition of one of embodiments 1 to 17, whereinsaid scintillator nanocrystal emits photon emission peaks within 350 nmto 400 nm.

Embodiment 21. The composition of one of embodiments 1 to 17, whereinsaid scintillator nanocrystal emits photon emission peaks within 350 nmto 370 nm.

Embodiment 22. The composition of one of embodiments 1 to 21, whereinsaid scintillator-activated photocleavable linker cleaves and releasessaid chemical agent upon absorbing photons from 100 nm to 600 nm.

Embodiment 23. The composition of one of embodiments 1 to 21, whereinsaid scintillator-activated photocleavable linker cleaves and releasessaid chemical agent upon absorbing photons from 350 nm to 400 nm.

Embodiment 24. The composition of one of embodiments 1 to 21, whereinsaid scintillator-activated photocleavable linker cleaves and releasessaid chemical agent upon absorbing photons from 350 nm to 370 nm.

Embodiment 25. The composition of one of embodiments 1 to 24, whereinsaid scintillator-activated photocleavable linker is covalently attachedto said chemical agent moiety and a surface of the scintillatornanocrystal.

Embodiment 26. The composition of embodiment 25, wherein said surface isa lipid bilayer surface or a silinated surface.

Embodiment 27. The composition of embodiment 25, wherein said surface isa silinated surface.

Embodiment 28. The composition of one of embodiments 1 to 27, whereinsaid scintillator-activated photocleavable linker has the formula:

wherein, L¹ and L² are independently bond, —C(O)—, —C(O)O—, —O—, —S—,—NH—, —C(O)NH—, —NHC(O)—, —S(O)₂—, —S(O)NH—, substituted orunsubstituted alkylene, substituted or unsubstituted heteroalkylene,substituted or unsubstituted cycloalkylene, substituted or unsubstitutedheterocycloalkylene, substituted or unsubstituted arylene, orsubstituted or unsubstituted heteroarylene; R¹ is independently halogen,—N₃, —CF₃, —CCl₃, —CBr₃, —CI₃, —CN, —COR², —OR², —NR²R³, —C(O)OR²,—C(O)NR²R³, —NO₂, —SR², —S(O)_(n1)R², —S(O)_(n1)OR², —S(O)_(n1)NR²R³,—NHNR²R³, —ONR²R³, —NHC(O)NHNR²R³, substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heterocycloalkyl, substitutedor unsubstituted aryl, or substituted or unsubstituted heteroaryl; R²and R³ are independently hydrogen, halogen, —N₃, —CF₃, —CCl₃, —CBr₃,—CI₃, —CN, —COH, —OH, —NH₂, —C(O)OH, —C(O)NH₂, —NO₂, —SH, —S(O)_(n1)H,—S(O)_(n2)OH, —S(O)_(n2)NH₂, —NHNH₂, —ONH₂, —NHC(O)NHNH₂, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, or substituted orunsubstituted heteroaryl; n is an integer from 0 to 4; n1 and n2 areindependently 1 or 2.

Embodiment 29. The composition of embodiment 28, wherein saidscintillator-activated photocleavable linker has the formula:

Embodiment 30. The composition of embodiment 28 or 29, wherein R¹ is—NO₂.

Embodiment 31. The composition of one of embodiments 28 to 30, whereinL² is —(CH₂)_(n3)—O—C(O)—, wherein n3 is an integer from 0 to 5.

Embodiment 32. The composition of embodiment 32, wherein n3 is 1.

Embodiment 33. The composition of one of embodiments 28 to 32, whereinL² is bound to the chemical agent moiety.

Embodiment 34. The composition of one of embodiments 1 to 33, whereinthe chemical agent moiety is covalently bound to saidscintillator-activated photocleavable linker through an amine group onsaid scintillator-activated photocleavable linker thereby forming an—NH-connecting moiety.

Embodiment 35. The composition of one of embodiments 1 to 34, whereinsaid chemical agent moiety is a drug moiety, hormone moiety, a metalmoiety, a radioprotective moiety, a cement moiety, a nucleotidetriphosphate moiety, a protein moiety, a polysaccharide moiety, aneurotransmitter moiety, an enzyme moiety, a tissue factor moiety or adetectable moiety.

Embodiment 36. The composition of embodiment 35, wherein said drugmoiety is an anticancer drug moiety or antibiotic drug moiety.

Embodiment 37. A method of delivering a chemical agent moiety to atarget site, said method comprising: (i) providing the composition ofone of embodiments 1 to 36 to a location at or near a target site, and(ii) cleaving said chemical agent from the remainder of said compound byexposing said composition to radiation thereby delivering said chemicalagent to said target site.

Embodiment 38. A method of delivering a chemical agent moiety to asubject, said method comprising: (i) administering the composition ofone of embodiments 1 to 36 to said subject, and (ii) cleaving saidchemical agent from the remainder of said compound by exposing saidcomposition to radiation thereby delivering said chemical agent to saidsubject.

Embodiment 39. The method of embodiment 38, wherein said subject is acancer patient and said chemical agent moiety is an anticancer drugagent, and wherein said composition is administered to said subject in atherapeutically effective amount.

What is claimed is:
 1. A composition comprising a scintillatornanocrystal, wherein the scintillator nanocrystal has a diameter fromabout 25 nm to about 300 nm, and wherein the scintillator nanocrystalcomprises (Y_(1-x)Pr_(x))₃Al₅O₁₂, where x is 0.0075, 0.01, 0.0125,0.015, or 0.0175.
 2. The composition of claim 1, wherein thescintillator nanocrystal further comprises a coating.
 3. The compositionof claim 1, wherein the scintillator nanocrystal further comprises achemical agent moiety, a scintillator nanocrystal-activatedphotocleavable linker, or a combination thereof.
 4. The composition ofclaim 1, wherein the scintillator nanocrystal further comprises achemical agent moiety and a scintillator nanocrystal-activatedphotocleavable linker; wherein the photocleavable linker operably linksthe scintillator nanocrystal and the chemical agent moiety.
 5. Thecomposition of claim 1, wherein the scintillator nanocrystal comprises ahost crystal lattice.
 6. The composition of claim 1, wherein thecomposition downconverts incident energy to emission energy that is lessthan the incident energy; wherein the incident energy comprises energythat is in the X-ray range, the gamma-ray range, or a combinationthereof; and wherein the emission energy comprises energy that is in theUV range.
 7. The composition of claim 1, wherein the scintillatornanocrystal emits at least one photon emission peak within 300 nm to 470nm.
 8. The composition of claim 4, wherein the scintillator-activatedphotocleavable linker cleaves and releases the chemical agent moietyupon absorbing photons from 100 nm to 600 nm.
 9. The composition ofclaim 4, wherein the scintillator-activated photocleavable linker iscovalently attached to a chemical agent moiety and a surface of thescintillator nanocrystal.
 10. The composition of claim 9, wherein thesurface is a lipid bilayer surface.
 11. The composition of claim 9,wherein the surface is a silinated surface.
 12. The composition of claim9, wherein said composition can be used to treat tumors.
 13. Thecomposition of claim 9, wherein said composition can be used to treat aninfection.
 14. The composition of claim 1, wherein the composition is ina powdered form.
 15. The composition of claim 1, wherein the compositionis in solid form or suspended in a liquid.
 16. The composition of claim1, wherein the scintillator nanocrystal produces two photon emissionpeaks between 300 nm and 400 nm when exposed to an X-ray of 50 keV. 17.The composition of claim 1, wherein the scintillator nanocrystalproduces a photon emission peak between 350 nm and 400 nm when exposedto an X-ray of 50 keV.
 18. The composition of claim 1, wherein thescintillator nanocrystals have a diameter of about 25 nm, about 50 nm,about 75 nm, about 100 nm, about 150 nm, about 200 nm, or a combinationthereof.
 19. The scintillator nanocrystal of claim 1, wherein thescintillator nanocrystal further comprises a silinated surface.
 20. Acomposition comprising a scintillator nanocrystal linked to a chemicalagent moiety through a scintillator-activated photocleavable linker;wherein (i) the scintillator nanocrystal comprises(Y_(1-x)Pr_(x))₃Al₅O₁₂, where x is 0.0075, 0.01, 0.0125, 0.015, or0.0175; wherein the scintillator nanocrystal has a diameter from about25 nm to about 300 nm; (ii) the chemical agent moiety comprises a drugmoiety, a hormone moiety, a metal moiety, a radioprotective moiety, acement moiety, a nucleotide triphosphate moiety, a protein moiety, apolysaccharide moiety, a neurotransmitter moiety, an enzyme moiety, atissue factor moiety, or a detectable moiety (ii) thescintillator-activated photocleavable linker has the formula:

wherein: L¹ and L² are independently bond, —C(O)—, —C(O)O—, —O—, —S—,—NH—, —C(O)NH—, —NHC(O)—, —S(O)₂—, —S(O)NH—, substituted orunsubstituted alkylene, substituted or unsubstituted heteroalkylene,substituted or unsubstituted cycloalkylene, substituted or unsubstitutedheterocycloalkylene, substituted or unsubstituted arylene, orsubstituted or unsubstituted heteroarylene; R¹ is independently halogen,—N₃, —CF₃, —CCl₃, —CBr₃, —CI₃, —CN, —COR², —OR², —NR²R³, —C(O)OR²,—C(O)NR²R³, —NO₂, —SR², —S(O)_(n1)R², —S(O)_(n1)OR², —S(O)_(n1)NR²R³,—NHNR²R³, —ONR²R³, —NHC(O)NHNR²R³, substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heterocycloalkyl, substitutedor unsubstituted aryl, or substituted or unsubstituted heteroaryl; R²and R³ are independently hydrogen, halogen, —N₃, —CF₃, —CCl₃, —CBr₃,—CI₃, —CN, —COH, —OH, —NH₂, —C(O)OH, —C(O)NH₂, —NO₂, —SH, —S(O)_(n1)H,—S(O)_(n2)OH, —S(O)_(n2)NH₂, —NHNH₂, —ONH₂, —NHC(O)NHNH₂, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, or substituted orunsubstituted heteroaryl; n is an integer from 0 to 4; and n1 and n2 areindependently 1 or
 2. 21. The composition of claim 20, wherein thechemical agent moiety is the drug moiety.
 22. The composition of claim21, wherein the drug moiety is to treat an infection or to treat cancer.